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1.
Commun Biol ; 4(1): 139, 2021 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514858

RESUMO

Leishmania infantum causes visceral leishmaniasis, a deadly vector-borne disease introduced to the Americas during the colonial era. This non-native trypanosomatid parasite has since established widespread transmission cycles using alternative vectors, and human infection has become a significant concern to public health, especially in Brazil. A multi-kilobase deletion was recently detected in Brazilian L. infantum genomes and is suggested to reduce susceptibility to the anti-leishmanial drug miltefosine. We show that deletion-carrying strains occur in at least 15 Brazilian states and describe diversity patterns suggesting that these derive from common ancestral mutants rather than from recurrent independent mutation events. We also show that the deleted locus and associated enzymatic activity is restored by hybridization with non-deletion type strains. Genetic exchange appears common in areas of secondary contact but also among closely related parasites. We examine demographic and ecological scenarios underlying this complex L. infantum population structure and discuss implications for disease control.

2.
PLoS Negl Trop Dis ; 14(10): e0008750, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33044986

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.

3.
Proc Natl Acad Sci U S A ; 117(40): 25159-25168, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32958676

RESUMO

The tropical Andes are an important natural laboratory to understand speciation in many taxa. Here we examined the evolutionary history of parasites of the Leishmania braziliensis species complex based on whole-genome sequencing of 67 isolates from 47 localities in Peru. We first show the origin of Andean Leishmania as a clade of near-clonal lineages that diverged from admixed Amazonian ancestors, accompanied by a significant reduction in genome diversity and large structural variations implicated in host-parasite interactions. Within the Andean species, patterns of population structure were strongly associated with biogeographical origin. Molecular clock and ecological niche modeling suggested that the history of diversification of the Andean lineages is limited to the Late Pleistocene and intimately associated with habitat contractions driven by climate change. These results suggest that changes in forestation over the past 150,000 y have influenced speciation and diversity of these Neotropical parasites. Second, genome-scale analyses provided evidence of meiotic-like recombination between Andean and Amazonian Leishmania species, resulting in full-genome hybrids. The mitochondrial genome of these hybrids consisted of homogeneous uniparental maxicircles, but minicircles originated from both parental species. We further show that mitochondrial minicircles-but not maxicircles-show a similar evolutionary pattern to the nuclear genome, suggesting that compatibility between nuclear-encoded mitochondrial genes and minicircle-encoded guide RNA genes is essential to maintain efficient respiration. By comparing full nuclear and mitochondrial genome ancestries, our data expand our appreciation on the genetic consequences of diversification and hybridization in parasitic protozoa.


Assuntos
Genoma Mitocondrial/genética , Interações Hospedeiro-Parasita/genética , Leishmania braziliensis/genética , Leishmaniose Cutânea/genética , Ecossistema , Florestas , Especiação Genética , Humanos , Leishmania braziliensis/patogenicidade , Leishmaniose Cutânea/epidemiologia , Leishmaniose Cutânea/parasitologia , Peru/epidemiologia , Filogeografia
4.
PLoS Negl Trop Dis ; 14(8): e0008509, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32804927

RESUMO

Leishmania species are responsible for a broad spectrum of diseases, denominated Leishmaniasis, affecting over 12 million people worldwide. During the last decade, there have been impressive efforts for sequencing the genome of most of the pathogenic Leishmania spp. as well as hundreds of strains, but large-scale proteomics analyses did not follow these achievements and the Leishmania proteome remained mostly uncharacterized. Here, we report a comprehensive comparative study of the proteomes of strains representing L. braziliensis, L. panamensis and L. guyanensis species. Proteins extracted by SDS-mediated lysis were processed following the multi-enzyme digestion-filter aided sample preparation (FASP) procedure and analysed by high accuracy mass spectrometry. "Total Protein Approach" and "Proteomic Ruler" were applied for absolute quantification of proteins. Principal component analysis demonstrated very high reproducibility among biological replicates and a very clear differentiation of the three species. Our dataset comprises near 7000 proteins, representing the most complete Leishmania proteome yet known, and provides a comprehensive quantitative picture of the proteomes of the three species in terms of protein concentration and copy numbers. Analysis of the abundance of proteins from the major energy metabolic processes allow us to highlight remarkably differences among the species and suggest that these parasites depend on distinct energy substrates to obtain ATP. Whereas L. braziliensis relies the more on glycolysis, L. panamensis and L. guyanensis seem to depend mainly on mitochondrial respiration. These results were confirmed by biochemical assays showing opposite profiles for glucose uptake and O2 consumption in these species. In addition, we provide quantitative data about different membrane proteins, transporters, and lipids, all of which contribute for significant species-specific differences and provide rich substrate for explore new molecules for diagnosing purposes. Data are available via ProteomeXchange with identifier PXD017696.


Assuntos
Leishmania/metabolismo , Proteínas de Protozoários/metabolismo , Regulação da Expressão Gênica/fisiologia , Glucose/metabolismo , Leishmania/genética , Consumo de Oxigênio , Proteômica , Proteínas de Protozoários/genética , Especificidade da Espécie
5.
PLoS Negl Trop Dis ; 14(7): e0007986, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32716941

RESUMO

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 , Filogenia
6.
Genes (Basel) ; 11(1)2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31861501

RESUMO

Pathogen fitness landscapes change when transmission cycles establish in non-native environments or spill over into new vectors and hosts. The introduction of Leishmania infantum in the Americas into the Neotropics during European colonization represents a unique case study to investigate the mechanisms of ecological adaptation of this important parasite. Defining the evolutionary trajectories that drive L. infantum fitness in this new environment are of great public health importance as they will allow unique insight into pathways of host/pathogen co-evolution and their consequences for region-specific changes in disease manifestation. This review summarizes current knowledge on L. infantum genetic and phenotypic diversity in the Americas and its possible role in the unique epidemiology of visceral leishmaniasis (VL) in the New World. We highlight the importance of appreciating adaptive molecular mechanisms in L. infantum to understand the parasites' successful establishment on the continent.


Assuntos
Leishmania infantum/classificação , Leishmaniose Visceral/transmissão , Oceano Atlântico , Evolução Molecular , Aptidão Genética , Humanos , Leishmania infantum/genética , Fenótipo
7.
PLoS Negl Trop Dis ; 13(12): e0007841, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31856199

RESUMO

BACKGROUND: Visceral Leishmaniasis (VL) is the most severe form of leishmaniasis because it can lead to death. In the Americas, 96% of cases are in Brazil, and despite efforts, the fatality rate has increased in the past years. We analyzed deaths associated to VL in Brazil and investigated the factors that could influence on the timeliness of fatal outcome with emphasis on time (tStoD). METHODOLOGY: The registered deaths by VL were sourced from the Brazilian National Notification System from 2007-2014. Through a retrospective cohort study, univariate and multivariable Cox proportional hazards model analysis were performed and investigated the factors that could influence the time (tStoD). These factors were analyzed through survival models. RESULTS: Out of the 1,589 reported deaths, the median for onset of the symptoms and the case notification date (tStoN) is 25 days (10-61), and for date of case notification and death (tNotD) is 9 days (4-17). The time (tStoN) to event investigation for HIV non-infected individuals was 1.4 (1.16-1.68) greater than the HIV positive group. At the same time peri-urban and urban area were 0.83 (0.47-1.44) and 1.33 (1.16-1.52), respectively. The explorations revealed apparent differences between the time to event investigation (both for tStoN and tNotD) and the age at the onset of the symptoms. According to the tStoN the rate of notification is 1.73 times greater in patients under 5 years old at the onset of the clinical symptoms compared to older patients. CONCLUSION: VL patients under 5 years old were diagnosed earlier and had shorter survival. It could mean that in younger population, although properly diagnosed, the fatality pattern might be related to the severity of the disease. Main host characteristics were evaluated, and age and co-infections seem to have an impact in the disease progression.


Assuntos
Leishmaniose Visceral/mortalidade , Mortalidade Prematura , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Brasil , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de Risco , Análise de Sobrevida , Adulto Jovem
8.
Sci Rep ; 9(1): 12962, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31506501

RESUMO

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/patologia
9.
Int J Parasitol ; 49(7): 555-567, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31108098

RESUMO

In the Indian subcontinent, infection with Leishmania donovani can cause fatal visceral leishmaniasis. Genetic variation in L. donovani is believed to occur rapidly from environmental changes and through selective drug pressures, thereby allowing continued disease occurrence in this region. All previous molecular markers that are commonly in use multilocus microsatellite typing and multilocus sequence typing, were monomorphic in L. donovani originating from the Indian subcontinent (with only a few exceptions) and hence are not suitable for this region. An multilocus sequence typing scheme consisting of a new set of seven housekeeping genes was developed in this study, based on recent findings from whole genome sequencing data. This new scheme was used to assess the genetic diversity amongst 22 autochthonous L. donovani isolates from Bangladesh. Nineteen additional isolates of the L. donovani complex (including sequences of L. donovani reference strain BPK282A1) from other countries were included for comparison. By using restriction fragment length polymorphism of the internal transcribed spacer 1 region (ITS1-RFLP) and ITS1 sequencing, all Bangladeshi isolates were confirmed to be L. donovani. Population genetic analyses of 41 isolates using the seven new MLST loci clearly separated L. donovani from Leishmania infantum. With this multilocus sequence typing scheme, seven genotypes were identified amongst Bangladeshi L. donovani isolates, and these isolates were found to be phylogenetically different compared with those from India, Nepal, Iraq and Africa. This novel multilocus sequence typing approach can detect intra- and inter-species variations within the L. donovani complex, but most importantly these molecular markers can be applied to resolve the phylogenetically very homogeneous L. donovani strains from the Indian subcontinent. Four of these markers were found suitable to differentiate strains originating from Bangladesh, with marker A2P being the most discriminative one.


Assuntos
Variação Genética , Leishmania donovani/classificação , Leishmania donovani/genética , Leishmaniose Visceral/parasitologia , Tipagem de Sequências Multilocus/métodos , Bangladesh , DNA Espaçador Ribossômico/genética , Genótipo , Humanos , Leishmania donovani/isolamento & purificação , Filogenia , Polimorfismo de Fragmento de Restrição , Sensibilidade e Especificidade
10.
Parasite ; 26: 30, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31120019

RESUMO

Tegumentary Leishmaniasis (TL) in the Brazilian Amazon region is associated with several Leishmania species. In this report, we describe two cases of TL related to Leishmania lindenbergi occurring in different locations of Rondônia state. After clinical diagnosis, lesion samples were collected for parasitological diagnoses via direct microscopic visualization, parasite isolation, and PCR. PCR reactions were positive in both clinical samples. Parasite isolation was possible for both patients, and isolates were submitted to species identification by isoenzyme electrophoresis and DNA sequencing. This report is the first to describe human infections caused by L. lindenbergi since the initial description and record of human infection by this species in 2002.


Assuntos
Leishmania/isolamento & purificação , Leishmaniose Cutânea/diagnóstico , Pele/patologia , Adulto , Brasil , Feminino , Humanos , Leishmania/classificação , Leishmania/genética , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Pele/parasitologia
11.
Artigo em Inglês | MEDLINE | ID: mdl-30533928

RESUMO

Leishmania (Viannia) braziliensis is the main etiological agent of tegumentary leishmaniasis in the neotropics. Here, we report a draft genome sequence (31.2 Mb) of an L. braziliensis strain from the western Amazon region of Brazil. This genome sequence will complement those available for other Leishmania species and contribute to further studies focusing on this parasite and the neglected diseases associated with it.

12.
mBio ; 9(6)2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30401775

RESUMO

Protozoan parasites of the genus Leishmania adapt to environmental change through chromosome and gene copy number variations. Only little is known about external or intrinsic factors that govern Leishmania genomic adaptation. Here, by conducting longitudinal genome analyses of 10 new Leishmania clinical isolates, we uncovered important differences in gene copy number among genetically highly related strains and revealed gain and loss of gene copies as potential drivers of long-term environmental adaptation in the field. In contrast, chromosome rather than gene amplification was associated with short-term environmental adaptation to in vitro culture. Karyotypic solutions were highly reproducible but unique for a given strain, suggesting that chromosome amplification is under positive selection and dependent on species- and strain-specific intrinsic factors. We revealed a progressive increase in read depth towards the chromosome ends for various Leishmania isolates, which may represent a nonclassical mechanism of telomere maintenance that can preserve integrity of chromosome ends during selection for fast in vitro growth. Together our data draw a complex picture of Leishmania genomic adaptation in the field and in culture, which is driven by a combination of intrinsic genetic factors that generate strain-specific phenotypic variations, which are under environmental selection and allow for fitness gain.IMPORTANCE Protozoan parasites of the genus Leishmania cause severe human and veterinary diseases worldwide, termed leishmaniases. A hallmark of Leishmania biology is its capacity to adapt to a variety of unpredictable fluctuations inside its human host, notably pharmacological interventions, thus, causing drug resistance. Here we investigated mechanisms of environmental adaptation using a comparative genomics approach by sequencing 10 new clinical isolates of the L. donovani, L. major, and L. tropica complexes that were sampled across eight distinct geographical regions. Our data provide new evidence that parasites adapt to environmental change in the field and in culture through a combination of chromosome and gene amplification that likely causes phenotypic variation and drives parasite fitness gains in response to environmental constraints. This novel form of gene expression regulation through genomic change compensates for the absence of classical transcriptional control in these early-branching eukaryotes and opens new venues for biomarker discovery.


Assuntos
Adaptação Fisiológica/genética , Dosagem de Genes , Genoma de Protozoário , Cariótipo , Leishmania donovani/genética , Telômero/genética , Animais , Cromossomos/genética , Cricetinae/parasitologia , Variações do Número de Cópias de DNA , Cães/parasitologia , Evolução Molecular , Amplificação de Genes , Regulação da Expressão Gênica , Genes de Protozoários , Aptidão Genética , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leishmania donovani/crescimento & desenvolvimento , Leishmaniose Visceral/parasitologia
13.
PLoS One ; 13(6): e0198727, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29912912

RESUMO

Cutaneous leishmaniasis is a neglected parasitic disease that manifests in infected individuals under different phenotypes, with a range of factors contributing to its broad clinical spectrum. One factor, Leishmania RNA Virus 1 (LRV1), has been described as an endosymbiont present in different species of Leishmania. LRV1 significantly worsens the lesion, exacerbating the immune response in both experimentally infected animals and infected individuals. Little is known about the composition and genetic diversity of these viruses. Here, we investigated the relationship between the genetic composition of LRV1 detected in strains of Leishmania (Viannia) braziliensis and L. (V.) guyanensis and the interaction between the endosymbiont and the parasitic species, analyzing an approximately 850 base pair region of the viral genome. We also included one LRV1 sequence detected in L. (V.) shawi, representing the first report of LRV1 in a species other than L. braziliensis and L. guyanensis. The results illustrate the genetic diversity of the LRV1 strains analyzed here, with smaller divergences detected among viral sequences from the same parasite species. Phylogenetic analyses showed that the LRV1 sequences are grouped according to the parasite species and possibly according to the population of the parasite in which the virus was detected, corroborating the hypothesis of joint evolution of the viruses with the speciation of Leishmania parasites.


Assuntos
Leishmania/virologia , Leishmaniavirus/genética , Coevolução Biológica/genética , Variação Genética/genética , Genoma Viral/genética , Leishmania braziliensis/virologia , Leishmania guyanensis/virologia , Leishmaniose/parasitologia , Filogenia , Análise de Sequência de DNA , América do Sul , Especificidade da Espécie
14.
PLoS Negl Trop Dis ; 12(4): e0006445, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29677186

RESUMO

The spleen is one of the main affected organs in canine visceral leishmaniasis (CVL). Disorganization of the splenic white pulp (SWP) has been associated with immunosuppression and disease progression. This study aims to assess structural and cellular changes in the splenic extracellular matrix of dogs with CVL, correlating these changes with the parasite load and clinical signs. Splenic fragments were collected from 41 naturally infected animals for parasite load quantification by quantitative PCR, histopathological analysis and immunohistochemistry for CD3+, CD4+, and CD8+ T cells; CD21+ B cells; Ki-67+, IFN-γ+, and IL-10+ cells; and the MMP-9 and ADAM-10 enzymes. Laminin, collagen and fibronectin deposition were also evaluated. The animals were grouped according to the level of SWP organization. SWP disorganization was accompanied by a reduction in the quantity of lymphoid follicles/mm2 (p > 0.0001). Animals with moderate to intense SWP disorganization showed more clinical signs (p = 0.021), higher laminin (p = 0.045) and collagen deposition (p = 0.036), higher MMP-9 expression (p = 0.035) and lower numbers of CD4+ T cells (p = 0.027) in the spleen than the animals with organized SWP. These data suggest that splenic structure and function are drastically altered and compromised during CVL.


Assuntos
Doenças do Cão/patologia , Matriz Extracelular/patologia , Leishmania infantum/fisiologia , Leishmaniose Visceral/veterinária , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Doenças do Cão/imunologia , Doenças do Cão/parasitologia , Cães , Matriz Extracelular/imunologia , Matriz Extracelular/parasitologia , Imuno-Histoquímica/veterinária , Leishmania infantum/imunologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/patologia , Linfócitos/imunologia , Linfócitos/patologia , Carga Parasitária/veterinária , Baço/imunologia , Baço/parasitologia , Baço/patologia
15.
Acta Trop ; 184: 29-37, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28965842

RESUMO

Cutaneous leishmaniasis (CL) is spread worldwide and is the most common manifestation of leishmaniasis. Diagnosis is performed by combining clinical and epidemiological features, and through the detection of Leishmania parasites (or DNA) in tissue specimens or trough parasite isolation in culture medium. Diagnosis of CL is challenging, reflecting the pleomorphic clinical manifestations of this disease. Skin lesions vary in severity, clinical appearance, and duration, and in some cases, they can be indistinguishable from lesions related to other diseases. Over the past few decades, PCR-based methods, including real-time PCR assays, have been developed for Leishmania detection, quantification and species identification, improving the molecular diagnosis of CL. This review provides an overview of many real-time PCR methods reported for the diagnostic evaluation of CL and some recommendations for the application of these methods for quantification purposes for clinical management and epidemiological studies. Furthermore, the use of real-time PCR for Leishmania species identification is also presented. The advantages of real-time PCR protocols are numerous, including increased sensitivity and specificity and simpler standardization of diagnostic procedures. However, despite the numerous assays described, there is still no consensus regarding the methods employed. Furthermore, the analytical and clinical validation of CL molecular diagnosis has not followed international guidelines so far. A consensus methodology comprising a DNA extraction protocol with an exogenous quality control and an internal reference to normalize parasite load is still needed. In addition, the analytical and clinical performance of any consensus methodology must be accurately assessed. This review shows that a standardization initiative is essential to guide researchers and clinical laboratories towards the achievement of a robust and reproducible methodology, which will permit further evaluation of parasite load as a surrogate marker of prognosis and monitoring of aetiological treatment, particularly in multi-centric observational studies and clinical trials.


Assuntos
DNA de Protozoário/genética , Leishmania/genética , Leishmania/isolamento & purificação , Leishmaniose Cutânea/diagnóstico , Carga Parasitária/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Humanos , Sensibilidade e Especificidade
18.
Int J Parasitol ; 47(10-11): 667-674, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28668326

RESUMO

This study explores the present day distribution of Lutzomyia longipalpis in relation to climate, and transfers the knowledge gained to likely future climatic conditions to predict changes in the species' potential distribution. We used ecological niche models calibrated based on occurrences of the species complex from across its known geographic range. Anticipated distributional changes varied by region, from stability to expansion or decline. Overall, models indicated no significant north-south expansion beyond present boundaries. However, some areas suitable both at present and in the future (e.g., Pacific coast of Ecuador and Peru) may offer opportunities for distributional expansion. Our models anticipated potential range expansion in southern Brazil and Argentina, but were variably successful in anticipating specific cases. The most significant climate-related change anticipated in the species' range was with regard to range continuity in the Amazon Basin, which is likely to increase in coming decades. Rather than making detailed forecasts of actual locations where Lu. longipalpis will appear in coming years, our models make interesting and potentially important predictions of broader-scale distributional tendencies that can inform heath policy and mitigation efforts.


Assuntos
Mudança Climática , Modelos Biológicos , Psychodidae/fisiologia , Distribuição Animal , Animais
19.
Rev Bras Parasitol Vet ; 25(4): 450-458, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27925060

RESUMO

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 Restrição
20.
Rev. bras. parasitol. vet ; 25(4): 450-458, Sept.-Dec. 2016. tab, graf
Artigo em Inglês | LILACS | ID: biblio-830049

RESUMO

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/parasitologia
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