RESUMO
High Resolution Melting Analysis (HRM) has been pointed out as a suitable alternative method to detect and identify Leishmania species. Herein, we aimed to evaluate the sensitivity, specificity, accuracy, and limitations of a HSP70-HRM protocol both as a diagnostic scheme applied in clinical samples and as a species typing tool for laboratory research and reference services. Our data reveal the pronounced species-typing potential of the HSP70-HRM in DNA from cultured parasites. For clinical samples, however, we advise caution due to parasite load-dependent accuracy. In light of these findings and considering the importance of parasite load determination for clinical and research purposes, we recommend the integration of the presented typing scheme and the previously published Leishmania quantifying approach as combined tools for clinicians, surveillance, and research.
Assuntos
Leishmania , Parasitos , Animais , Leishmania/genética , Proteínas de Choque Térmico HSP70/genéticaRESUMO
Leishmania parasites present astonishing adaptative abilities that represent a matter of life or death within disparate environments during the heteroxenous parasite life cycle. From an evolutionary perspective, organisms develop methods of overcoming such challenges. Strategies that extend beyond the genetic diversity have been discussed and include variability between parasite cells during the infections of their hosts. The occurrence of Leishmania subpopulation fluctuations with variable structural genomic contents demonstrates that a single strain might shelter the variability required to overcome inconsistent environments. Such intrastrain variability provides parasites with an extraordinary ability to adapt and thus survive and propagate. However, different perspectives on this evolution have been proposed. Strains or species living in the same environment can cooperate but also compete. These interactions might increase the replication rate of some parasites but cause the loss of more aggressive competitors for others. Adaptive responses to intra- and interspecific competition can evolve as a fixed strategy (replication is adapted to the average genetic complexity of infections) or an optional strategy (replication varies according to the genetic complexity of the current infection). This review highlights the complexity of interspecies and intrastrain interactions among Leishmania parasites as well as the different factors that influence this interplay.
RESUMO
Treatment of visceral leishmaniasis in Brazil still relies on meglumine antimoniate, with less than ideal efficacy and safety, making new therapeutic tools an urgent need. The oral drug miltefosine was assayed in a phase II clinical trial in Brazil with cure rates lower than previously demonstrated in India. The present study investigated the susceptibility to miltefosine in 73 Brazilian strains of Leishmania infantum from different geographic regions, using intracellular amastigote and promastigote assays. The EC50 for miltefosine of 13 of these strains evaluated in intracellular amastigotes varied between 1.41 and 4.57 µM. The EC50 of the 73 strains determined in promastigotes varied between 5.89 and 23.7 µM. No correlation between in vitro miltefosine susceptibility and the presence of the miltefosine sensitive locus was detected among the tested strains. The relatively low heterogeneity in miltefosine susceptibility observed for the 73 strains tested in this study suggests the absence of decreased susceptibility to miltefosine in Brazilian L. infantum and does not exclude future clinical evaluation of miltefosine for VL treatment in Brazil.
RESUMO
Leishmaniasis is a worldwide neglected disease, encompassing asymptomatic infections and different clinical forms, such as American Tegumentary Leishmaniasis (ATL) which is part of the complex of diseases caused by protozoan parasites from Leishmania genus, transmitted by sand fly vectors. As a neglected disease, much effort is still needed in treatment and diagnosis. Currently, ATL diagnosis is mainly made by parasite detection by microscopy. The sensitivity of the method varies, and factors such as collection procedures interfere. Molecular approaches, specially based on Real Time PCR (qPCR) technique, has been widely used to detect Leishmania infection and to quantify parasite load, once it is a simple, rapid and sensitive methodology, capable to detect low parasite concentrations and less prone to variability. Although many studies have been already published addressing the use of this technique, an improvement on these methodologies, including an analytical validation, standardization and data association is demanded. Moreover, a proper validation by the assay by the use of clinical samples is still required. In this sense, the purpose of the present work is to compare the performance of qPCR using two commonly used targets (18S rDNA and HSP70) with an internal control (RNAse P) in multiplex reactions. Additionally, we validated reactions by assaying 88 samples from patients presenting different clinical forms of leishmaniasis (cutaneous, mucosal, recent and old lesions), representing the diversity found in Brazil's Amazon Region. Following the methodology proposed herein, the results indicate the use of both qPCR assays, 18S rDNA and HSP70, to achieve a very good net sensitivity (98.5%) and specificity (100%), performing simultaneous or sequential testing, respectively. With this approach, our main goal is to conclude the first step of a further multicenter study to propose the standardization of detection and quantification of Leishmania.
Assuntos
DNA Ribossômico/genética , Proteínas de Choque Térmico HSP70/genética , Leishmania/isolamento & purificação , Leishmaniose Cutânea/parasitologia , Carga Parasitária/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , DNA de Protozoário/análise , DNA de Protozoário/genética , DNA Ribossômico/análise , Proteínas de Choque Térmico HSP70/análise , Humanos , Leishmaniose Cutânea/patologia , Sensibilidade e Especificidade , Pele/parasitologiaRESUMO
Genetic polymorphisms in natural Leishmania populations have been reported in endemic areas. Microsatellite typing is a useful tool to elucidate the genetic variability of parasite strains, due to its capability for high-resolution mapping of genomic targets. The present study employed multilocus microsatellite typing (MLMT) to explore the genotypic composition of Leishmania infantum in naturally infected dogs by genotyping parasites infecting different tissues with or without in vitro expansion. Eighty-six samples were collected from 46 animals in an endemic region of visceral leishmaniasis (VL). MLMT was performed for 38 spleen samples and 48 L. infantum cultures isolated from different tissues. Of the 86 samples, 23 were effectively genotyped by MLMT, identifying nine multilocus genotypes (MLG; referred to as MLG A-I). MLGs A, B and C were detected in more than one type of tissue and in more than one sample. Conversely, MLG D-I were uniquely detected in one sample each. The results showed that multiple genotype infections occur within a single host and tissue. Paired sample analysis revealed the presence of different MLMT alleles in 14 dogs, while the same MLG allele was present in 15 animals. STRUCTURE analysis demonstrated the presence of two populations; 13 samples displayed a similar admixture of both ancestral populations, and these were not assigned to any population. Only samples for which Q ≥ 0.70 after CLUMPP alignment were considered to be part of Population 1 (POP1) or Population 2 (POP2). POP2 comprised the majority of samples (n = 54) compared to POP1 (n = 19). This study presents evidence of multiple genotype infections (caused by L. infantum) in dogs in an area with high VL transmission. Further investigations must be undertaken to determine the effects of multiple infection on the host immune response and disease dynamics and treatment.
Assuntos
Doenças do Cão/parasitologia , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/veterinária , Animais , Cães , Feminino , Variação Genética , Genótipo , Leishmania infantum/classificação , Leishmania infantum/genética , Leishmaniose Visceral/parasitologia , Masculino , Repetições de Microssatélites , FilogeniaRESUMO
In canine visceral leishmaniasis (CVL), splenic white pulp (SWP) disorganization has been associated with disease progression, reduced cytokine and chemokine expression and failure to control the parasite load. This profile is compatible with the cellular exhaustion previously shown in human visceral leishmaniasis. The present study aimed to evaluate the in situ expression of cellular exhaustion markers and their relation to clinical signs, SWP disorganization and parasite load. Forty dogs naturally infected by Leishmania infantum were grouped according to levels of SWP organization and parasite load. SWP disorganization was associated with reductions in the periarteriolar lymphatic sheath and lymphoid follicles/mm2 and worsening of the disease. Apoptotic cells expressing CTLA-4+ increased in dogs with disorganized SWP and a high parasite load. In the same group, PD-L1 and LAG-3 gene expression were reduced. A higher number of CD21+TIM-3+ B cells was detected in disorganized spleens than in organized spleens. Apoptosis is involved in periarteriolar lymphatic sheath reduction and lymphoid follicle atrophy and is associated with CTLA-4+ cell reductions in the splenic tissue of dogs with visceral leishmaniasis (VL). Failure to control the parasite load was observed, suggesting that cell exhaustion followed by T and B cell apoptosis plays a role in the immunosuppression observed in CVL.
Assuntos
Biomarcadores/análise , Doenças do Cão/imunologia , Leishmania infantum/imunologia , Leishmaniose Visceral/veterinária , Carga Parasitária , Baço/imunologia , Baço/parasitologia , Animais , Citocinas/metabolismo , Doenças do Cão/parasitologia , Doenças do Cão/patologia , Cães , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/patologia , Baço/patologiaRESUMO
The sandfly Trichophoromyia auraensis has recently evolved as a proven vector of Leishmania (Viannia) endemic to state of Acre in the north of Brazil. This note is intended to propose a correction in the report of the first occurrence of natural infection of Leishmania (Viannia) in this species. We and the other scientific groups reinforced that Tr. auraensis is a possible vector involved in the transmission of American cutaneous leishmaniasis in Acre, Brazil.
Assuntos
Animais , Leishmaniose Cutânea/parasitologia , Insetos Vetores/classificação , Insetos Vetores/parasitologia , Leishmania/isolamento & purificação , Psychodidae , Psychodidae/parasitologia , BrasilRESUMO
Studies on the sandfly fauna to evaluate natural infection indexes are still limited in the Brazilian Amazon, a region with an increasing incidence of cutaneous leishmaniasis. Here, by using a multiplex polymerase chain reaction directed to Leishmania kDNA and hybridisation, we were able to identify L. (Viannia) subgenus in 12 out of 173 sandflies captured in the municipality of Rio Branco, Acre state, revealing a positivity of 6.94%. By sequencing the Leishmania 234 bp-hsp70 amplified products from positive samples, infection by L. (V.) braziliensis was confirmed in five sandflies: one Evandromyia saulensis, three Trichophoromyia auraensis and one Pressatia sp. The finding of L. (Viannia) DNA in two Ev. saulensis corresponds to the first record of possible infection associated with this sandfly. Moreover, our study reveals for the first time in Brazil, Th. auraensis and Pressatia sp. infected by L. (Viannia) parasites.
Assuntos
Insetos Vetores/parasitologia , Leishmania/classificação , Leishmaniose Cutânea/transmissão , Psychodidae/parasitologia , Animais , Brasil , DNA de Protozoário/genética , Insetos Vetores/classificação , Leishmania/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex , Psychodidae/classificaçãoRESUMO
Studies on the sandfly fauna to evaluate natural infection indexes are still limited in the Brazilian Amazon, a region with an increasing incidence of cutaneous leishmaniasis. Here, by using a multiplex polymerase chain reaction directed to Leishmania kDNA and hybridisation, we were able to identify L. (Viannia) subgenus in 12 out of 173 sandflies captured in the municipality of Rio Branco, Acre state, revealing a positivity of 6.94%. By sequencing the Leishmania 234 bp-hsp70 amplified products from positive samples, infection by L. (V.) braziliensis was confirmed in five sandflies: one Evandromyia saulensis, three Trichophoromyia auraensis and one Pressatia sp. The finding of L. (Viannia) DNA in two Ev. saulensis corresponds to the first record of possible infection associated with this sandfly. Moreover, our study reveals for the first time in Brazil, Th. auraensis and Pressatia sp. infected by L. (Viannia) parasites.
Assuntos
Animais , Psychodidae/classificação , Psychodidae/parasitologia , Leishmaniose Cutânea/transmissão , Insetos Vetores/classificação , Insetos Vetores/parasitologia , Leishmania/isolamento & purificação , Leishmania/classificação , Brasil , Reação em Cadeia da Polimerase MultiplexRESUMO
This study describes the occurrence of dogs naturally co-infected with Hepatozoon canis and two Leishmania species: L. infantum or L. braziliensis. Four dogs serologically diagnosed with Visceral Leishmaniasis were euthanized. Liver and spleen samples were collected for histopathological analysis and DNA isolation. H. canis meronts were observed in tissues from all four dogs. H. canis infection was confirmed by PCR followed by sequencing of a fragment of 18S rRNA gene. Leishmania detection and typing was confirmed by ITS1' PCR-RFLP and parasite burden was calculated using ssrRNA quantitative qPCR. A DPP - Dual Path platform test was performed. One out (Dog #2) of four animals was asymptomatic. Dogs #1 and #4 were infected by L. infantum and were DPP test positive. Dogs #2 and #3 were infected by L. braziliensis and were DPP test negative. Furthermore, visceral dissemination was observed in Dogs #2 and #3, since L. braziliensis was detected in liver and spleen samples. The visceral dissemination of L. braziliensis associated with systemic signs suggested that this co-infection could influence the parasite burden and disease progression.
Assuntos
Coccidiose/veterinária , Coinfecção/veterinária , Doenças do Cão/parasitologia , Leishmaniose Visceral/veterinária , Animais , Coccídios , Coccidiose/parasitologia , Coinfecção/parasitologia , Cães , Leishmania infantum , Leishmaniose Visceral/parasitologia , Polimorfismo de Fragmento de RestriçãoRESUMO
Abstract This study describes the occurrence of dogs naturally co-infected with Hepatozoon canis and two Leishmania species: L. infantum or L. braziliensis. Four dogs serologically diagnosed with Visceral Leishmaniasis were euthanized. Liver and spleen samples were collected for histopathological analysis and DNA isolation. H. canis meronts were observed in tissues from all four dogs. H. canis infection was confirmed by PCR followed by sequencing of a fragment of 18S rRNA gene. Leishmania detection and typing was confirmed by ITS1' PCR-RFLP and parasite burden was calculated using ssrRNA quantitative qPCR. A DPP - Dual Path platform test was performed. One out (Dog /2) of four animals was asymptomatic. Dogs /1 and /4 were infected by L. infantum and were DPP test positive. Dogs /2 and /3 were infected by L. braziliensis and were DPP test negative. Furthermore, visceral dissemination was observed in Dogs /2 and /3, since L. braziliensis was detected in liver and spleen samples. The visceral dissemination of L. braziliensis associated with systemic signs suggested that this co-infection could influence the parasite burden and disease progression.(AU)
Resumo O presente estudo descreve a ocorrência de coinfecção com Hepatozoon canis e duas espécies de Leishmania (L. infantum ou L. braziliensis) em cães. Quatro cães sorologicamente diagnosticados com leishmaniose visceral foram eutanasiados. Amostras do baço e fígado foram submetidas à histopatologia e extração de DNA. Merontes de H. canis foram observados nos quatro cães. A infecção por H. canis foi confirmada por PCR e sequenciamento de um fragmento do gene 18S rRNA. A infecção por Leishmania e tipagem foram realizadas por PCR-RFLP do região intergênica ITS1. A carga parasitária foi calculada pela qPCR quantitativa baseada no gene ssrRNA. O teste DPP - Dual Path platform foi realizado. Apenas o Cão /2 era assintomático. Os cães /1 e /4 estavam infectados com L. infantum e foram positivos no DPP. Os cães /2 e /3 estavam infectados com L. braziliensis e foram negativos no DPP. Além disso, visceralização foi observada nos cães /2 e /3, nos quais L. braziliensis foi detectada em amostras de baço e fígado. A visceralização da L. braziliensis associada a sinais clínicos sistêmicos sugerem que esta coinfecção pode ter influenciado na carga parasitária e progressão da doença.(AU)
Assuntos
Animais , Cães , Coccídios , Coccidiose/parasitologia , Coccidiose/veterinária , Coinfecção/parasitologia , Coinfecção/veterinária , Doenças do Cão/parasitologia , Leishmania infantum , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/veterinária , Polimorfismo de Fragmento de RestriçãoRESUMO
Abstract This study describes the occurrence of dogs naturally co-infected with Hepatozoon canis and two Leishmania species: L. infantum or L. braziliensis. Four dogs serologically diagnosed with Visceral Leishmaniasis were euthanized. Liver and spleen samples were collected for histopathological analysis and DNA isolation. H. canis meronts were observed in tissues from all four dogs. H. canis infection was confirmed by PCR followed by sequencing of a fragment of 18S rRNA gene. Leishmania detection and typing was confirmed by ITS1' PCR-RFLP and parasite burden was calculated using ssrRNA quantitative qPCR. A DPP - Dual Path platform test was performed. One out (Dog #2) of four animals was asymptomatic. Dogs #1 and #4 were infected by L. infantum and were DPP test positive. Dogs #2 and #3 were infected by L. braziliensis and were DPP test negative. Furthermore, visceral dissemination was observed in Dogs #2 and #3, since L. braziliensis was detected in liver and spleen samples. The visceral dissemination of L. braziliensis associated with systemic signs suggested that this co-infection could influence the parasite burden and disease progression.
Resumo O presente estudo descreve a ocorrência de coinfecção com Hepatozoon canis e duas espécies de Leishmania (L. infantum ou L. braziliensis) em cães. Quatro cães sorologicamente diagnosticados com leishmaniose visceral foram eutanasiados. Amostras do baço e fígado foram submetidas à histopatologia e extração de DNA. Merontes de H. canis foram observados nos quatro cães. A infecção por H. canis foi confirmada por PCR e sequenciamento de um fragmento do gene 18S rRNA. A infecção por Leishmania e tipagem foram realizadas por PCR-RFLP do região intergênica ITS1. A carga parasitária foi calculada pela qPCR quantitativa baseada no gene ssrRNA. O teste DPP - Dual Path platform foi realizado. Apenas o Cão #2 era assintomático. Os cães #1 e #4 estavam infectados com L. infantum e foram positivos no DPP. Os cães #2 e #3 estavam infectados com L. braziliensis e foram negativos no DPP. Além disso, visceralização foi observada nos cães #2 e #3, nos quais L. braziliensis foi detectada em amostras de baço e fígado. A visceralização da L. braziliensis associada a sinais clínicos sistêmicos sugerem que esta coinfecção pode ter influenciado na carga parasitária e progressão da doença.
Assuntos
Animais , Cães , Coccidiose/veterinária , Doenças do Cão/parasitologia , Coinfecção/veterinária , Leishmaniose Visceral/veterinária , Polimorfismo de Fragmento de Restrição , Coccídios , Coccidiose/parasitologia , Leishmania infantum , Coinfecção/parasitologia , Leishmaniose Visceral/parasitologiaRESUMO
INTRODUCTION: The aim of this study was quantify annexin A1 expression in macrophages and cluster of differentiation 4 (CD4) + and cluster of differentiation 8 (CD8)+ T cells from the skin of patients with cutaneous leishmaniasis (n=55) and correlate with histopathological aspects. METHODS: Infecting species were identified by polymerase chain reaction-restriction fragment length polymorphism, and expression of annexin A1 was analyzed by immunofluorescence. RESULTS: All patients (n = 55) were infected with Leishmania braziliensis . Annexin A1 was expressed more abundantly in CD163 + macrophages in infected skin (p < 0.0001) than in uninfected skin. In addition, macrophages in necrotic exudative reaction lesions expressed annexin A1 at higher levels than those observed in granulomatous (p < 0.01) and cellular lesions p < 0.05). This difference might be due to the need to clear both parasites and necrotic tissue from necrotic lesions. CD4 + cells in cellular lesions expressed annexin A1 more abundantly than did those in necrotic (p < 0.05) and granulomatous lesions (p < 0.01). Expression in CD8 + T cells followed the same trend. These differences might be due to the pervasiveness of lymphohistiocytic and plasmacytic infiltrate in cellular lesions. CONCLUSIONS: Annexin A1 is differentially expressed in CD163 + macrophages and T cells depending on the histopathological features of Leishmania -infected skin, which might affect cell activation.
Assuntos
Anexina A1/metabolismo , Leishmania/classificação , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/patologia , Anexina A1/análise , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Estudos Transversais , Feminino , Imunofluorescência , Humanos , Leishmaniose Cutânea/parasitologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de RestriçãoRESUMO
BACKGROUND: The main transmission route of Leishmania infantum is through the bites of sand flies. However, alternative mechanisms are being investigated, such as through the bites of ticks, which could have epidemiological relevance. The objective of this work was to verify the presence of Leishmania spp. in Rhipicephalus sanguineus sensu lato collected from naturally infected dogs in the Federal District of Brazil. METHODS: Ticks were dissected to remove their intestines and salivary glands for DNA extraction and the subsequent amplification of the conserved region of 120 bp of kDNA and 234 bp of the hsp70 gene of Leishmania spp. The amplified kDNA products were digested with endonucleases HaeIII and BstUI and were submitted to DNA sequencing. Isolated Leishmania parasites from these ticks were analyzed by multilocus enzyme electrophoresis, and the DNA obtained from this culture was subjected to microsatellite analyses. RESULTS: Overall, 130 specimens of R. sanguineus were collected from 27 dogs. Leishmania spp. were successfully isolated in culture from five pools of salivary glands and the intestines of ticks collected from four dogs. The amplified kDNA products from the dog blood samples and from the tick cultures, when digested by HaeIII and BstUI, revealed the presence of L. braziliensis and L. infantum. One strain was cultivated and characterized as L. infantum by enzyme electrophoresis. The amplified kDNA products from the blood of one dog showed a sequence homology with L. braziliensis; however, the amplified kDNA from the ticks collected from this dog showed a sequence homology to L. infantum. CONCLUSION: The results confirm that the specimens of R. sanguineus that feed on dogs naturally infected by L. infantum contain the parasite DNA in their intestines and salivary glands, and viable L. infantum can be successfully isolated from these ectoparasites.
Assuntos
Vetores Aracnídeos/parasitologia , Doenças do Cão/parasitologia , Leishmania infantum/isolamento & purificação , Rhipicephalus sanguineus/parasitologia , Infestações por Carrapato/veterinária , Animais , Doenças do Cão/epidemiologia , Cães , Infestações por Carrapato/epidemiologia , Infestações por Carrapato/parasitologiaRESUMO
ABSTRACTINTRODUCTION:The aim of this study was quantify annexin A1 expression in macrophages and cluster of differentiation 4 (CD4) + and cluster of differentiation 8 (CD8)+ T cells from the skin of patients with cutaneous leishmaniasis (n=55) and correlate with histopathological aspects.METHODS:Infecting species were identified by polymerase chain reaction-restriction fragment length polymorphism, and expression of annexin A1 was analyzed by immunofluorescence.RESULTS:All patients (n = 55) were infected with Leishmania braziliensis . Annexin A1 was expressed more abundantly in CD163 + macrophages in infected skin (p < 0.0001) than in uninfected skin. In addition, macrophages in necrotic exudative reaction lesions expressed annexin A1 at higher levels than those observed in granulomatous (p < 0.01) and cellular lesions p < 0.05). This difference might be due to the need to clear both parasites and necrotic tissue from necrotic lesions. CD4 + cells in cellular lesions expressed annexin A1 more abundantly than did those in necrotic (p < 0.05) and granulomatous lesions (p < 0.01). Expression in CD8 + T cells followed the same trend. These differences might be due to the pervasiveness of lymphohistiocytic and plasmacytic infiltrate in cellular lesions.CONCLUSIONS:Annexin A1 is differentially expressed in CD163 + macrophages and T cells depending on the histopathological features of Leishmania -infected skin, which might affect cell activation.
Assuntos
Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Anexina A1/metabolismo , Leishmania/classificação , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/patologia , Anexina A1/análise , Estudos Transversais , Imunofluorescência , Leishmaniose Cutânea/parasitologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de RestriçãoRESUMO
Métodos moleculares estão sendo cada vez mais empregados para diagnóstico, estudos taxonômicos, filogenéticos e epidemiológicos envolvendo este parasita. [...] O principal objetivo deste trabalho foi desenvolver marcadores multilocus para Leishmania (Viannia) e avaliar sua aplicabilidade em estudos epidemiológicos. Os objetivos específicos incluíram: i) o desenvolvimento de um painel multi locus sequence analisys (MLSA) para cepas de Leishmania (Viannia) que circulam no Brasil que permita, concomitantemente, a identificação de isolados e a realização de estudos filogenéticos; ii) o estudo da estrutura da população de cepas de Leishmania (Viannia) quanto à clonalidade e ocorrência de eventos de recombinação pela determinação de perfis de microssatélites; iii) avaliação da aplicabilidade do painel MLSA como ferramenta epidemiológica para as leishmanioses; iv) a descrição de eventos moleculares em leishmânia que podem interferir nos resultados obtidos a partir de marcadores moleculares empregados...
Molecular methods have been often applied for diagnosis, studies oftaxonomy, phylogeny and epidemiology. [...] The main objective ofthis study was to develop multi locus markers for Leishmania (Viannia) and to evaluate its applicability in epidemiological studies. The specific objectives were: i) to develop a multi locus sequence analyses (MLSA) panel with Leishmania (Viannia) strains from Brazil that allows the species identification and phylogenetic inferences to be performed; ii) to execute a population structure study through microsatellites profiles (MLMT); iii) to evaluate the MLSA panel as anepidemiological tool; iv) to describe molecular events that may interfere in the results obtained by the molecular markers employed. After MLSA and MLMT, the results could be compared. MLMT defined two main populations, one comprising L. (V.) guyanensis and other L. (V.) braziliensis strainsfrom the Atlantic coast; recombination signs were detected for both. A third group, quite heterogeneous, included L. (V.) braziliensis strains from northern Brazil and the other species L. (V.)shawi, L. (V.) naiffi, and L. (V.) lainson...
Assuntos
Humanos , Leishmania/classificação , Leishmania/genética , Leishmaniose Cutânea/epidemiologia , Epidemiologia MolecularRESUMO
BACKGROUND/OBJECTIVES: Parasites of the subgenus Leishmania (Viannia) cause varying clinical symptoms ranging from cutaneous leishmaniases (CL) with single or few lesions, disseminated CL (DL) with multiple lesions to disfiguring forms of mucocutaneous leishmaniasis (MCL). In this population genetics study, 37 strains of L. (V.) guyanensis, 63 of L. (V.) braziliensis, four of L. (V.) shawi, six of L. (V.) lainsoni, seven of L. (V.) naiffi, one each of L. (V.) utingensis and L. (V.) lindenbergi, and one L. (V.) lainsoni/L. naiffi hybrid from different endemic foci in Brazil were examined for variation at 15 hyper-variable microsatellite markers. METHODOLOGY/PRINCIPAL FINDINGS: The multilocus microsatellite profiles obtained for the 120 strains were analysed using both model- and distance-based methods. Significant genetic diversity was observed for all L. (Viannia) strains studied. The two cluster analysis approaches identified two principal genetic groups or populations, one consisting of strains of L. (V.) guyanensis from the Amazon region and the other of strains of L. (V.) braziliensis isolated along the Atlantic coast of Brazil. A third group comprised a heterogeneous assembly of species, including other strains of L. braziliensis isolated from the north of Brazil, which were extremely polymorphic. The latter strains seemed to be more closely related to those of L. (V.) shawi, L. (V.) naiffi, and L. (V.) lainsoni, also isolated in northern Brazilian foci. The MLMT approach identified an epidemic clone consisting of 13 strains of L. braziliensis from Minas Gerais, but evidence for recombination was obtained for the populations of L. (V.) braziliensis from the Atlantic coast and for L. (V.) guyanensis. CONCLUSIONS/SIGNIFICANCE: Different levels of recombination versus clonality seem to occur within the subgenus L. (Viannia). Though clearly departing from panmixia, sporadic, but long-term sustained recombination might explain the tremendous genetic diversity and limited population structure found for such L. (Viannia) strains.
Assuntos
Variação Genética , Leishmania/classificação , Leishmania/genética , Recombinação Genética , Brasil , Análise por Conglomerados , DNA de Protozoário/genética , Genótipo , Técnicas de Genotipagem , Humanos , Leishmania/isolamento & purificação , Leishmaniose/parasitologia , Repetições de MicrossatélitesRESUMO
As the distribution of Candida species and their susceptibility to antifungal agents have changed, a new means of accurately and rapidly identifying these species is necessary for the successful early resolution of infection and the subsequent reduction of morbidity and mortality. The current work aimed to evaluate ribosomal RNA gene sequencing for the identification of medically relevant Candida species in comparison with a standard phenotypic method. Eighteen reference strains (RSs), 69 phenotypically identified isolates and 20 inconclusively identified isolates were examined. Internal transcribed spaces (ITSs) and D1/D2 of the 26S ribosomal RNA gene regions were used as targets for sequencing. Additionally, the sequences of the ITS regions were used to establish evolutionary relationships. The sequencing of the ITS regions was successful for 88% (94/107) of the RS and isolates, whereas 100% of the remaining 12% (13/107) of the samples were successfully analysed by sequencing the D1/D2 region. Similarly, genotypic analysis identified all of the RS and isolates, including the 20 isolates that were not phenotypically identified. Phenotypic analysis, however, misidentified 10% (7/69) of the isolates. Phylogenetic analysis allowed the confirmation of the relationships between evolutionarily close species. Currently, the use of genotypic methods is necessary for the correct identification of Candida species.
Assuntos
Candida/genética , DNA Fúngico/análise , DNA Espaçador Ribossômico/genética , Genes de RNAr/genética , Candida/classificação , Genótipo , Humanos , Fenótipo , Reação em Cadeia da Polimerase , Análise de Sequência de RNARESUMO
As the distribution of Candida species and their susceptibility to antifungal agents have changed, a new means of accurately and rapidly identifying these species is necessary for the successful early resolution of infection and the subsequent reduction of morbidity and mortality. The current work aimed to evaluate ribosomal RNA gene sequencing for the identification of medically relevant Candida species in comparison with a standard phenotypic method. Eighteen reference strains (RSs), 69 phenotypically identified isolates and 20 inconclusively identified isolates were examined. Internal transcribed spaces (ITSs) and D1/D2 of the 26S ribosomal RNA gene regions were used as targets for sequencing. Additionally, the sequences of the ITS regions were used to establish evolutionary relationships. The sequencing of the ITS regions was successful for 88% (94/107) of the RS and isolates, whereas 100% of the remaining 12% (13/107) of the samples were successfully analysed by sequencing the D1/D2 region. Similarly, genotypic analysis identified all of the RS and isolates, including the 20 isolates that were not phenotypically identified. Phenotypic analysis, however, misidentified 10% (7/69) of the isolates. Phylogenetic analysis allowed the confirmation of the relationships between evolutionarily close species. Currently, the use of genotypic methods is necessary for the correct identification of Candida species.
Assuntos
Humanos , Candida/genética , DNA Fúngico/análise , DNA Espaçador Ribossômico/genética , Genes de RNAr/genética , Candida/classificação , Genótipo , Fenótipo , Reação em Cadeia da Polimerase , Análise de Sequência de RNARESUMO
In this study, PCR assays targeting different Leishmania heat-shock protein 70 gene (hsp70) regions, producing fragments ranging in size from 230-390 bp were developed and evaluated to determine their potential as a tool for the specific molecular diagnosis of cutaneous leishmaniasis (CL). A total of 70 Leishmania strains were analysed, including seven reference strains (RS) and 63 previously typed strains. Analysis of the RS indicated a specific region of 234 bp in the hsp70 gene as a valid target that was highly sensitive for detection of Leishmania species DNA with capacity of distinguishing all analyzed species, after polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). This PCR assay was compared with other PCR targets used for the molecular diagnosis of leishmaniasis: hsp70 (1400-bp region), internal transcribed spacer (ITS)1 and glucose-6-phosphate dehydrogenase (G6pd). A good agreement among the methods was observed concerning the Leishmania species identification. Moreover, to evaluate the potential for molecular diagnosis, we compared the PCR targets hsp70-234 bp, ITS1, G6pd and mkDNA using a panel of 99 DNA samples from tissue fragments collected from patients with confirmed CL. Both PCR-hsp70-234 bp and PCR-ITS1 detected Leishmania DNA in more than 70% of the samples. However, using hsp70-234 bp PCR-RFLP, identification of all of the Leishmania species associated with CL in Brazil can be achieved employing a simpler and cheaper electrophoresis protocol.