Genotypic profiles of Leishmania (Viannia) braziliensis strains from cutaneous leishmaniasis patients and their relationship with the response to meglumine antimoniate treatment: a pilot study.

BACKGROUND: Forty-four strains isolated from a cohort of cutaneous leishmaniasis (CL) patients who did or did not respond to one course of treatment with meglumine antimoniate were investigated to explore genetic polymorphisms in parasite kinetoplast DNA minicircles. Leishmania (Viannia) braziliensis strains isolated from responder (R) and non-responder (NR) patients who acquired infection in Rio de Janeiro or in other Brazilian states were studied using low-stringency single-specific primer polymerase chain reaction (LSSP-PCR) to identify genetic polymorphisms. RESULTS: Polymorphisms were observed in parasites recovered from patient lesions. No association was found between a specific genotype and R or NR patients. Phenetic analysis grouped the genotypes into three main clusters, with similarity indices varying from 0.72 to 1.00. Although no specific genotype association was detected, at least one group of L. (V.) braziliensis genotypes that circulates in Rio de Janeiro was discriminated in clusters I and III, showing phenotypes of good and poor responses to treatment, respectively. Cluster I comprised parasite profiles recovered from R patients from Rio de Janeiro and in cluster III, NR samples were prevalent. Cluster II comprised 24 isolates, with 21 from Rio de Janeiro and three from other states, equally distributed between R and NR patients. Additionally, we found that parasites sharing all common genetic characteristics acted differently in response to treatment. CONCLUSIONS: These results are of clinical-epidemiological importance since they demonstrate that populations of L. (V.) braziliensis that exhibit high levels of genetic similarity also display different phenotypes associated with meglumine antimoniate responses in cutaneous leishmaniasis patients.

Higher sensitivity of immunohistochemistry for bona fide diagnosis of dog Leishmania (Viannia) braziliensis-driven American tegumentary leishmaniasis: description of an optimized immunohistochemistry method.

BACKGROUND: The in situ detection of parasite antigens in tissue sections by immunohistochemistry (IHC) is a diagnostic alternative for human American tegumentary leishmaniasis (ATL), but has not been used for the diagnosis of cutaneous lesions in dogs with ATL. This study describes the results of IHC for the detection of amastigote forms and other Leishmania sp. antigen-positive cells and compares the results of IHC, histopathology and cytopathology for the diagnosis of canine ATL. In addition, possible cross-reactivity with sporotrichosis is analyzed. METHODS: Forty paraffin-embedded biopsies and 40 smears of cutaneous lesions from dogs with ATL, confirmed by isolation and characterization of Leishmania (Viannia) braziliensis, and 40 paraffin-embedded biopsies of cutaneous lesions from dogs with sporotrichosis, confirmed by isolation of Sporothrix schenckii in culture (control group), were studied. RESULTS: Immunohistochemistry was more sensitive in detecting amastigote forms than cytopathology and histopathology, with a positivity rate of 70% (n=28) versus 37.5% and 22.5% for histopathology and cytopathology, respectively. Cytoplasmic staining of mononuclear and endothelial cells was detected by IHC, which was highly specific since no cytoplasmic staining of these cells or staining of fungal structures was observed in sporotrichosis fragments. CONCLUSIONS: In view of the higher sensitivity of IHC in detecting Leishmania sp. antigen and patterns of positivity for Leishmania sp. antigen compared to histopathology or cytopathology and the absence of cross-reactions with sporotrichosis, we recommend this technique for the diagnosis of canine tegumentary leishmaniasis.

Molecular study of Trypanosoma caninum isolates based on different genetic markers.

Trypanosoma caninum is a parasite recently described in dogs, whose life cycle is rather unknown. Here, we performed a genetic study with T. caninum samples obtained in different Brazilian regions. The study was based on PCR assays target to small and large subunit ribosomal DNA (rDNA) (18S rDNA and 24Sα rDNA), cytochrome B (Cyt b), and internal transcribed spacer 1 rDNA (ITS1 rDNA) following by the sequence analysis. Additionally, we used primers for the variable regions of kinetoplast DNA (kDNA) minicircles and endonucleases restriction in the ITS1 rDNA amplification product. T. caninum samples displayed the same patterns. Tree construction confirmed the close relationship between T. caninum samples, regardless of the molecular target used and endonuclease restriction digestion revealed that all samples have the same restriction profile. Therefore, T. caninum seems to be a genetically homogeneous specie. In the kDNA assay, T. caninum possessed a different molecular size profile with respect to others trypanosomes, 330 and 350 bp. This study provides nucleotide sequences from different regions of the genome of T. caninum that certainly facilitate future studies.

PCR associated with molecular hybridization detects Leishmania (Viannia) braziliensis in healthy skin in canine tegumentary leishmaniasis.

Tegumentary leishmaniasis (TL) is a zoonotic disease that affects humans and domestic dogs. In Brazil, TL is considered endemic, and Leishmania (Viannia) braziliensis is the prevalent species causing this disease. There is debate about the role of dogs (Canis familiaris) as domestic reservoirs in the transmission cycle of TL. To date, classical parasitological techniques, including parasite isolation in culture media, have been able to detect parasites only from cutaneous lesions. In this study, we detected L. (V.) braziliensis DNA in intact skin fragments collected from 3 naturally infected dogs from the state of Rio de Janeiro, Brazil, with the use of PCR techniques associated with molecular hybridization. The detection of parasitic DNA in this anatomical site is an important finding vis-à-vis the importance of the domestic dogs in endemic areas of TL.

Leishmania (Leishmania)hertigi in a porcupine (Coendou sp.) found in Brasília, Federal District, Brazil

Studies report the occurrence of Leishmania(Leishmania) hertigi in northern states of Brazil. In the present investigation, we describe the isolation ofL. (L.) hertigi from a porcupine (Coendou sp.) found in Brasília, Federal District, center-west region of Brazil. During a study on canine visceral leishmaniasis conducted in the city of Brasília, Federal District, a porcupine was found dead on a public road. The animal was identified and fragments of intact skin and spleen were collected for isolation of parasite in the culture. This report of the occurrence of L. hertigi in another part of Brazil may help establish the distribution of this parasite in the country. Further studies are needed to better understand the role of L. hertigi in the pathology and pathogenesis of leishmaniasis and its survival in mammals and possible vectors.

Alguns estudos relatam o encontro de Leishmania(Leishmania) hertigi em estados da região Norte do Brasil. Na presente investigação, os autores descrevem o isolamento deLeishmania (Leishmania)hertigi em um porco-espinho (Coendou sp.) encontrado em Brasília, Distrito Federal, região Centro-oeste do Brasil. Durante um estudo de leishmaniose visceral canina conduzido na cidade de Brasília, Distrito Federal, um porco-espinho foi encontrado morto em via pública. O animal foi identificado e fragmentos de pele íntegra e baço foram coletados para isolamento do parasito em cultura. Esse relato da ocorrência de L. hertigi em outra região do Brasil pode contribuir para evidenciar a distribuição desse parasita no país. Vale ressaltar a necessidade de estudos para compreensão do papel L. hertigi na patologia e patogênese da leishmaniose e a sua sobrevivência em mamíferos e possíveis vetores.

First autochthonous case of canine visceral leishmaniasis in Volta Redonda, Rio de Janeiro, Brazil

In Brazil, American visceral leishmaniasis (AVL) is caused byLeishmania (Leishmania) chagasi and its main vector isLutzomyia longipalpis. Cases of canine visceral leishmaniasis (CVL) in non-endemic areas have been reported over the last few years throughout the country. The objective of this research note is to describe an autochthonous case of CVL that occurred in the municipality of Volta Redonda, state of Rio de Janeiro, an area where the disease is not endemic, alerting veterinarians and the scientific community to the expansion of this important zoonosis and advising veterinary practitioners on how to deal with a suspicion of CVL. Canine visceral leishmaniasis can be misdiagnosed within a broad spectrum of canine diseases based on clinical and laboratory findings. Therefore, knowledge of its clinical manifestations, specific and sensitive laboratory diagnostic tests and parasitological procedures are of the utmost importance for rapid confirmation and notification of a case, thus contributing directly to the control of a focus.

No Brasil a leishmaniose visceral americana (LVA) é causada porLeishmania (Leishmania) chagasi e tem como seu principal vetor Lutzomyia longipalpis. Nos últimos anos vêm sendo relatados casos de leishmaniose visceral canina (LVC) em áreas não endêmicas em todo país. O objetivo desta nota é descrever um caso autóctone de LVC no município de Volta Redonda, Estado do Rio de Janeiro, área não endêmica para essa doença e assim, chamar a atenção dos clínicos veterinários e da comunidade científica para a expansão dessa importante zoonose, além de orientar os médicos veterinários, como proceder frente a um caso suspeito de LVC. A LVC pode ser clínica e laboratorialmente confundida com uma ampla gama de patologias caninas e o conhecimento de suas manifestações clínicas e de procedimentos laboratoriais específicos e sensíveis para esse diagnóstico, são de grande importância para uma rápida confirmação e notificação do caso, contribuindo assim diretamente para o controle do foco.

Leishmania (Leishmania)hertigi in a porcupine (Coendou sp.) found in Brasília, Federal District, Brazil

Studies report the occurrence of Leishmania(Leishmania) hertigi in northern states of Brazil. In the present investigation, we describe the isolation ofL. (L.) hertigi from a porcupine (Coendou sp.) found in Brasília, Federal District, center-west region of Brazil. During a study on canine visceral leishmaniasis conducted in the city of Brasília, Federal District, a porcupine was found dead on a public road. The animal was identified and fragments of intact skin and spleen were collected for isolation of parasite in the culture. This report of the occurrence of L. hertigi in another part of Brazil may help establish the distribution of this parasite in the country. Further studies are needed to better understand the role of L. hertigi in the pathology and pathogenesis of leishmaniasis and its survival in mammals and possible vectors.

Alguns estudos relatam o encontro de Leishmania(Leishmania) hertigi em estados da região Norte do Brasil. Na presente investigação, os autores descrevem o isolamento deLeishmania (Leishmania)hertigi em um porco-espinho (Coendou sp.) encontrado em Brasília, Distrito Federal, região Centro-oeste do Brasil. Durante um estudo de leishmaniose visceral canina conduzido na cidade de Brasília, Distrito Federal, um porco-espinho foi encontrado morto em via pública. O animal foi identificado e fragmentos de pele íntegra e baço foram coletados para isolamento do parasito em cultura. Esse relato da ocorrência de L. hertigi em outra região do Brasil pode contribuir para evidenciar a distribuição desse parasita no país. Vale ressaltar a necessidade de estudos para compreensão do papel L. hertigi na patologia e patogênese da leishmaniose e a sua sobrevivência em mamíferos e possíveis vetores.

Leishmaniose visceral canina: caso alóctone no município de Resende, estado do Rio de Janeiro, Brasil

A leishmaniose visceral (LV) é uma endemia em franca expansão geográfica. Este relato apresenta um caso de importação de LV canina para o município de Resende, estado do Rio de Janeiro, onde não havia até o momento registro de casos humanos ou caninos da parasitose. O animal era oriundo do estado de Minas Gerais, área endêmica de LV, e apresentou clínica compatível com o processo patológico em questão. O diagnóstico foi realizado por meio de avaliação sorológica com o teste rápido imunocromatográfico DPP® (DUAL PATH PLATFORM) e teste de ELISA, ambos com resultado positivo, além de cultura parasitológica em Meio NNN acrescido de meio Schneider a partir de punções de medula óssea e linfonodos, biópsias de pele íntegra, lesões cutâneas, linfonodo e baço, obtendo-se o isolamento de formas promastigotas compatíveis com Leishmania spp., com posterior caracterização de Leishmania (Leishmania) chagasi através de eletroforese de isoenzimas. O levantamento entomológico com utilização de armadilhas luminosas do tipo CDC, realizado no peridomicílio três vezes por semana durante três meses, não evidenciou a presença de flebotomíneos, e o inquérito sorológico canino, procedido em 144 animais ao redor da residência do caso positivo de LV, descartou novos casos da parasitose por meio do teste rápido imunocromatográfico DPP® e/ou confirmação por ELISA. Os resultados e o histórico de deslocamen

Leishmaniose visceral canina: caso alóctone no município de Resende, estado do Rio de Janeiro, Brasil

A leishmaniose visceral (LV) é uma endemia em franca expansão geográfica. Este relato apresenta um caso de importação de LV canina para o município de Resende, estado do Rio de Janeiro, onde não havia até o momento registro de casos humanos ou caninos da parasitose. O animal era oriundo do estado de Minas Gerais, área endêmica de LV, e apresentou clínica compatível com o processo patológico em questão. O diagnóstico foi realizado por meio de avaliação sorológica com o teste rápido imunocromatográfico DPP® (DUAL PATH PLATFORM) e teste de ELISA, ambos com resultado positivo, além de cultura parasitológica em Meio NNN acrescido de meio Schneider a partir de punções de medula óssea e linfonodos, biópsias de pele íntegra, lesões cutâneas, linfonodo e baço, obtendo-se o isolamento de formas promastigotas compatíveis com Leishmania spp., com posterior caracterização de Leishmania (Leishmania) chagasi através de eletroforese de isoenzimas. O levantamento entomológico com utilização de armadilhas luminosas do tipo CDC, realizado no peridomicílio três vezes por semana durante três meses, não evidenciou a presença de flebotomíneos, e o inquérito sorológico canino, procedido em 144 animais ao redor da residência do caso positivo de LV, descartou novos casos da parasitose por meio do teste rápido imunocromatográfico DPP® e/ou confirmação por ELISA. Os resultados e o histórico de deslocamen

Participation of heparin binding proteins from the surface of Leishmania (Viannia) braziliensis promastigotes in the adhesion of parasites to Lutzomyia longipalpis cells (Lulo) in vitro.

BACKGROUND: Leishmania (V.) braziliensis is a causative agent of cutaneous leishmaniasis in Brazil. During the parasite life cycle, the promastigotes adhere to the gut of sandflies, to avoid being eliminated with the dejection. The Lulo cell line, derived from Lutzomyia longipalpis (Diptera: Psychodidae), is a suitable in vitro study model to understand the features of parasite adhesion. Here, we analyze the role of glycosaminoglycans (GAGs) from Lulo cells and proteins from the parasites in this event. METHODS: Flagellar (Ff) and membrane (Mf) fractions from promastigotes were obtained by differential centrifugation and the purity of fractions confirmed by western blot assays, using specific antibodies for cellular compartments. Heparin-binding proteins (HBP) were isolated from both fractions using a HiTrap-Heparin column. In addition, binding of promastigotes to Lulo cells or to a heparin-coated surface was assessed by inhibition assays or surface plasmon resonance (SPR) analysis. RESULTS: The success of promastigotes subcellular fractionation led to the obtainment of Ff and Mf proteins, both of which presented two main protein bands (65.0 and 55.0 kDa) with affinity to heparin. The contribution of HBPs in the adherence of promastigotes to Lulo cells was assessed through competition assays, using HS or the purified HBPs fractions. All tested samples presented a measurable inhibition rate when compared to control adhesion rate (17 ± 2.0% of culture cells with adhered parasites): 30% (for HS 20 µg/ml) and 16% (for HS 10 µg/ml); HBP Mf (35.2% for 10 µg/ml and 25.4% for 20 µg/ml) and HBP Ff (10.0% for 10 µg/ml and 31.4% for 20 µg/ml). Additionally, to verify the presence of sulfated GAGs in Lulo cells surface and intracellular compartment, metabolic labeling with radioactive sulfate was performed, indicating the presence of an HS and chondroitin sulfate in both cell sections. The SPR analysis performed further confirmed the presence of GAGs ligands on L. (V.) braziliensis promastigote surfaces. CONCLUSIONS: The data presented here point to evidences that HBPs present on the surface of L. (V.) braziliensis promastigotes participate in adhesion of these parasites to Lulo cells through HS participation.

Leishmania (Viannia) braziliensis: insights on subcellular distribution and biochemical properties of heparin-binding proteins.

Leishmaniasis is a vector-borne disease and an important public health issue. Glycosaminoglycan ligands in Leishmania parasites are potential targets for new strategies to control this disease. We report the subcellular distribution of heparin-binding proteins (HBPs) in Leishmania (Viannia) braziliensis and specific biochemical characteristics of L. (V.) braziliensis HBPs. Promastigotes were fractionated, and flagella and membrane samples were applied to HiTrap Heparin affinity chromatography columns. Heparin-bound fractions from flagella and membrane samples were designated HBP Ff and HBP Mf, respectively. Fraction HBP Ff presented a higher concentration of HBPs relative to HBP Mf, and SDS-PAGE analyses showed 2 major protein bands in both fractions (65 and 55 kDa). The 65 kDa band showed gelatinolytic activity and was sensitive to inhibition by 1,10-phenanthroline. The localization of HBPs on the promastigote surfaces was confirmed using surface plasmon resonance (SPR) biosensor analysis by binding the parasites to a heparin-coated sensor chip; that was inhibited in a dose-dependent manner by pre-incubating the parasites with variable concentrations of heparin, thus indicating distinct heparin-binding capacities for the two fractions. In conclusion, protein fractions isolated from either the flagella or membranes of L. (V.) braziliensis promastigotes have characteristics of metallo-proteinases and are able to bind to glycosaminoglycans.

Canine visceral leishmaniasis: seroprevalence and risk factors in Cuiabá, Mato Grosso, Brazil

In Brazil, canine visceral leishmaniasis (CVL) is endemic and the number of cases in humans and dogs has increased in the Midwest region. A transversal study was carried out in endemic areas from Cuiabá, State of Mato Grosso, to assess data on seroprevalence and risk factors associated to canine infection. Four hundred and thirty (430) dogs were randomly evaluated through indirect fluorescence antibody test (IFAT) considering variables related to the animals, the environment and the knowledge by owners on CVL aspects and control. From 430 dogs, 95 (22.1%) were seroreagent for leishmaniasis and animals living in rural environments present risk 1.9 times higher for acquiring the disease than those in urban environments (p = 0.01; OR 1.9). Factors related to animals' habits, such as free access to the street and guard function were considered indicators to predict infection by Leishmania sp. (p 0.05) by statistical univariate analysis. The presence of agricultural activities was also a fact that contributed for the insurgence of the infection (p = 0.02; OR 1.68). The results contributed to the knowledge on the aspects of CVL in Cuiabá and point to an urgent need to include educational and sanitary programs in the city, since the region presents favorable characteristics for spreading the infection of CVL as already observed in other Brazilian cities.

No Brasil, a leishmaniose visceral canina (CLV) é endêmica e, na região Centro-Oeste, o número de casos em humanos e cães tem aumentado. Um estudo transversal foi realizado em áreas endêmicas de Cuiabá (MT) com objetivo de avaliar dados sobre a soroprevalência e determinar os fatores de risco associados à infecção canina. Quatrocentos e trinta (430) cães foram aleatoriamente avaliados pelo teste de imunofluorescência indireta, considerando-se variáveis relacionadas aos animais, o ambiente e o conhecimento por parte dos proprietários sobre aspectos da CLV e seu controle. Dos 430 cães, 95 (22,1%) apresentaram-se soros reagentes para leishmaniose, e os animais que viviam em ambiente rural apresentaram risco 1,9 vezes maior de adquirir a infecção dos que aqueles em ambiente urbano (p = 0,01; OR 1,9). Fatores relacionados aos hábitos dos animais, tais como o livre acesso à rua e função de guarda, foram considerados indicadores para prever a infecção por Leishmania sp. (p. 0,05) em análise estatística univariada. A presença de atividade agrícola foi também um fato que contribuiu para a ocorrência da infecção (p = 0,02; OR 1,68). Os resultados contribuem para o conhecimento sobre os aspectos da CVL em Cuiabá e apontam para uma necessidade urgente de incluir ações educativas e sanitárias na cidade, já que a região possui características favoráveis para a dispersão da doença como já observado em outras cidades.

Canine visceral leishmaniasis: seroprevalence and risk factors in Cuiabá, Mato Grosso, Brazil

In Brazil, canine visceral leishmaniasis (CVL) is endemic and the number of cases in humans and dogs has increased in the Midwest region. A transversal study was carried out in endemic areas from Cuiabá, State of Mato Grosso, to assess data on seroprevalence and risk factors associated to canine infection. Four hundred and thirty (430) dogs were randomly evaluated through indirect fluorescence antibody test (IFAT) considering variables related to the animals, the environment and the knowledge by owners on CVL aspects and control. From 430 dogs, 95 (22.1%) were seroreagent for leishmaniasis and animals living in rural environments present risk 1.9 times higher for acquiring the disease than those in urban environments (p = 0.01; OR 1.9). Factors related to animals' habits, such as free access to the street and guard function were considered indicators to predict infection by Leishmania sp. (p 0.05) by statistical univariate analysis. The presence of agricultural activities was also a fact that contributed for the insurgence of the infection (p = 0.02; OR 1.68). The results contributed to the knowledge on the aspects of CVL in Cuiabá and point to an urgent need to include educational and sanitary programs in the city, since the region presents favorable characteristics for spreading the infection of CVL as already observed in other Brazilian cities.

No Brasil, a leishmaniose visceral canina (CLV) é endêmica e, na região Centro-Oeste, o número de casos em humanos e cães tem aumentado. Um estudo transversal foi realizado em áreas endêmicas de Cuiabá (MT) com objetivo de avaliar dados sobre a soroprevalência e determinar os fatores de risco associados à infecção canina. Quatrocentos e trinta (430) cães foram aleatoriamente avaliados pelo teste de imunofluorescência indireta, considerando-se variáveis relacionadas aos animais, o ambiente e o conhecimento por parte dos proprietários sobre aspectos da CLV e seu controle. Dos 430 cães, 95 (22,1%) apresentaram-se soros reagentes para leishmaniose, e os animais que viviam em ambiente rural apresentaram risco 1,9 vezes maior de adquirir a infecção dos que aqueles em ambiente urbano (p = 0,01; OR 1,9). Fatores relacionados aos hábitos dos animais, tais como o livre acesso à rua e função de guarda, foram considerados indicadores para prever a infecção por Leishmania sp. (p. 0,05) em análise estatística univariada. A presença de atividade agrícola foi também um fato que contribuiu para a ocorrência da infecção (p = 0,02; OR 1,68). Os resultados contribuem para o conhecimento sobre os aspectos da CVL em Cuiabá e apontam para uma necessidade urgente de incluir ações educativas e sanitárias na cidade, já que a região possui características favoráveis para a dispersão da doença como já observado em outras cidades.

Canine visceral leishmaniasis: two autochthonous cases in florianópolis municipality, santa catarina state, Brazil / Leishmaniose Visceral Canina: Dois casos autóctones no município de Florianópolis, estado de Santa Catarina

Background: Brazil faces an expansion and urbanization of American visceral leishmaniasis with human and canine cases in several metropolises. This report describes two cases of autochthonous canine visceral leishmaniasis in the city of Florianópolis, state of Santa Catarina, classified as an area previously without disease transmission.Case: Five dogs from Canto dos Araçás, Lagoa da Conceição, Florianópolis municipality, which had no history of displacement to other municipalities, were studied by collecting clinical specimens in an attempt to isolate the parasite and to confirm the infection by Leishmania. For sampling the animals were sedated and after local shaving, asepsis and anesthesia with lidocaine HCl 2%, two fragments of apparently healthy skin of the scapular region with the aid of a 3 mm punch were obtained. The two fragments were placed in sterile saline plus antibiotics and antifungal agents solution to attempt the isolation of the parasite in NNN culture medium plus Schneiders medium. In addition to the collection of skin fragment, animals underwent bone marrow (BM) needle aspiration, obtained from the manubrium of the sternum, using a 20 mL syringe with 40x12 mm needle and aspiration biopsy of popliteal lymph nodes using Valeri citoaspirador 10 mL syringe and 25x8 mm needle, both performed after local shaving, asepsis and anesthesia with 2% lidocaine. The mate

Background: Brazil faces an expansion and urbanization of American visceral leishmaniasis with human and canine cases in several metropolises. This report describes two cases of autochthonous canine visceral leishmaniasis in the city of Florianópolis, state of Santa Catarina, classified as an area previously without disease transmission.Case: Five dogs from Canto dos Araçás, Lagoa da Conceição, Florianópolis municipality, which had no history of displacement to other municipalities, were studied by collecting clinical specimens in an attempt to isolate the parasite and to confirm the infection by Leishmania. For sampling the animals were sedated and after local shaving, asepsis and anesthesia with lidocaine HCl 2%, two fragments of apparently healthy skin of the scapular region with the aid of a 3 mm punch were obtained. The two fragments were placed in sterile saline plus antibiotics and antifungal agents solution to attempt the isolation of the parasite in NNN culture medium plus Schneiders medium. In addition to the collection of skin fragment, animals underwent bone marrow (BM) needle aspiration, obtained from the manubrium of the sternum, using a 20 mL syringe with 40x12 mm needle and aspiration biopsy of popliteal lymph nodes using Valeri citoaspirador 10 mL syringe and 25x8 mm needle, both performed after local shaving, asepsis and anesthesia with 2% lidocaine. The mate

Canine visceral leishmaniasis: two autochthonous cases in florianópolis municipality, santa catarina state, Brazil / Leishmaniose Visceral Canina: Dois casos autóctones no município de Florianópolis, estado de Santa Catarina

Background: Brazil faces an expansion and urbanization of American visceral leishmaniasis with human and canine cases in several metropolises. This report describes two cases of autochthonous canine visceral leishmaniasis in the city of Florianópolis, state of Santa Catarina, classified as an area previously without disease transmission.Case: Five dogs from Canto dos Araçás, Lagoa da Conceição, Florianópolis municipality, which had no history of displacement to other municipalities, were studied by collecting clinical specimens in an attempt to isolate the parasite and to confirm the infection by Leishmania. For sampling the animals were sedated and after local shaving, asepsis and anesthesia with lidocaine HCl 2%, two fragments of apparently healthy skin of the scapular region with the aid of a 3 mm punch were obtained. The two fragments were placed in sterile saline plus antibiotics and antifungal agents solution to attempt the isolation of the parasite in NNN culture medium plus Schneiders medium. In addition to the collection of skin fragment, animals underwent bone marrow (BM) needle aspiration, obtained from the manubrium of the sternum, using a 20 mL syringe with 40x12 mm needle and aspiration biopsy of popliteal lymph nodes using Valeri citoaspirador 10 mL syringe and 25x8 mm needle, both performed after local shaving, asepsis and anesthesia with 2% lidocaine. The mate

Background: Brazil faces an expansion and urbanization of American visceral leishmaniasis with human and canine cases in several metropolises. This report describes two cases of autochthonous canine visceral leishmaniasis in the city of Florianópolis, state of Santa Catarina, classified as an area previously without disease transmission.Case: Five dogs from Canto dos Araçás, Lagoa da Conceição, Florianópolis municipality, which had no history of displacement to other municipalities, were studied by collecting clinical specimens in an attempt to isolate the parasite and to confirm the infection by Leishmania. For sampling the animals were sedated and after local shaving, asepsis and anesthesia with lidocaine HCl 2%, two fragments of apparently healthy skin of the scapular region with the aid of a 3 mm punch were obtained. The two fragments were placed in sterile saline plus antibiotics and antifungal agents solution to attempt the isolation of the parasite in NNN culture medium plus Schneiders medium. In addition to the collection of skin fragment, animals underwent bone marrow (BM) needle aspiration, obtained from the manubrium of the sternum, using a 20 mL syringe with 40x12 mm needle and aspiration biopsy of popliteal lymph nodes using Valeri citoaspirador 10 mL syringe and 25x8 mm needle, both performed after local shaving, asepsis and anesthesia with 2% lidocaine. The mate

Comparison of the sensitivity of imprint and scraping techniques in the diagnosis of American tegumentary leishmaniasis in a referral centre in Rio de Janeiro, Brazil.

American tegumentary leishmaniasis (ATL) is an infectious disease that presents a wide spectrum of clinical manifestations making parasitological tests important for its diagnosis. Direct examination, although considered of low sensitivity is still employed mainly in areas with poor laboratory infrastructure. The aim of this study was to standardize the method of collecting and reading the scraping procedure and to then compare sensitivity of this procedure on two sites of the lesion (outer edge-OE and inner edge-IE) and of the imprint against the reference method (isolation in culture) in a group of 110 patients treated at a Referral Center in Rio de Janeiro, Brazil. ATL diagnosis was confirmed in 40 patients (36.4%), 39 cases were caused by L. braziliensis and 1 by L. amazonensis. Imprint was positive in 28 patients and scraping in OE in 17 and in IE in 25 patients, resulting in sensitivity of 70%, 42.5%, and 62.5% respectively. When the three direct examinations were combined, sensitivity value attained 77.5%. Aspects related to ease and quality of the collected material, pain intensity and frequency of bleeding in the scraping procedure were also broached and discussed in this study. The parameters of accuracy presented indicate that the direct methods can be safely used in ATL diagnosis, principally in IE scraping, as it is easy to produce and the examination is not costly, which allows the procedure to be repeated at different moments which, in turn, increases the possibility of finding the parasite. Despite that the direct methods are technically widespread, they are not standardized and the parameters of accuracy are unknown. If we consider the high incidence of leishmaniasis in low-income areas, the implantation of standardized and selective methods would provide advances in the diagnosis of leishmaniasis.

Immunoenzymatic assay for the diagnosis of American tegumentary leishmaniasis using soluble and membrane-enriched fractions from infectious Leishmania (Viannia) braziliensis.

The diagnosis of American tegumentary leishmaniasis (ATL) is based on the visualization or isolation of the parasite, which is a time-consuming and poorly sensitive method. In this study, we evaluated the accuracy and reliability of ELISA for the diagnosis of ATL using soluble (SF) and membrane-enriched (MF) antigen fractions obtained from an infectious strain of Leishmania (Viannia) braziliensis. A total of 152 serum samples investigated at a referral center in Rio de Janeiro, Brazil, between 2005 and 2007 were studied. Each sample was tested twice with each fraction for the calculation of reliability (intraclass coefficient (ICC)). Cut-off values of 0.22 (SF) and 0.33 (MF) were defined. The use of the fractions resulted in good discrimination between patients, with a large area under the curve (P<0.0001), but no difference was observed between the two fractions (P=0.45). Sensitivity was 89.5% for each fraction, specificity was 89.5% for SF and 93.4% for MF, and the positive likelihood ratio was 8.5 for SF and 13.6 for MF. The ICCs were excellent (SF: 0.96 and MF: 0.90). The antigens tested provided precision and accuracy for the diagnosis of ATL, with SF being recommended due to its lower cost and greater practicality.

Use of ELISA employing Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi antigens for the detection of IgG and IgG1 and IgG2 subclasses in the diagnosis of American tegumentary leishmaniasis in dogs.

American tegumentary leishmaniasis (ATL) shows a reduced humoral response in dogs and levels of specific antibodies may therefore not be detected by indirect immunofluorescence. Although the sensitivity of enzyme-linked immunosorbent assay (ELISA) is higher than that of indirect immunofluorescence, the best antigen for the diagnosis of ATL in dogs has not been defined. The detection of IgG subclasses represents an alternative to increase the efficiency of the serological diagnosis. In Rio de Janeiro, sporotrichosis is the main differential diagnosis of ATL in dogs, and a sensitive, specific and little invasive method that permits the discrimination of the two diseases is desired. In the present study, 69 serum samples, 34 obtained from dogs with ATL and 35 from dogs with sporotrichosis, all of them with a confirmed etiological diagnosis, were tested. The samples were analyzed by ELISA using Leishmania (Viannia) braziliensis and L. (L.) chagasi antigens for the detection of anti-Leishmania IgG, IgG1 and IgG2. The use of L. (V.) braziliensis antigens for the detection of IgG and IgG2 yielded the best results. Using L. (L.) chagasi antigen, the sensitivity and specificity for the detection of IgG were 82.4% and 100%, respectively, whereas both sensitivity and specificity were 97.1% with the L. (V.) braziliensis antigen. No improvement in the performance of the test was observed when IgG2 was analyzed separately. The IgG1 assays presented low accuracy, irrespective of the antigen used: sensitivity and specificity of 58.8% and 60% for L. (V.) braziliensis and of 64.7% and 77.1% for L. (L.) chagasi, respectively. The present results suggest that IgG ELISA using the L. (V.) braziliensis shows the best performance for the diagnosis of ATL, permitting the discrimination between cases of ATL and sporotrichosis in dogs.

Post mortem parasitological evaluation of dogs seroreactive for Leishmania from Rio de Janeiro, Brazil.

A parasitological study was conducted on 66 dogs seroreactive for Leishmania captured as a control measure of visceral leishmaniasis in the State of Rio de Janeiro, Brazil. Biological samples from different anatomical sites were collected during autopsy of the animals and cultured on biphasic medium (NNN/Schneider). The Leishmania isolates were characterized by isoenzyme electrophoresis. Leishmania was isolated from 80.3% of the animals: 12 animals with Leishmania (Viannia) braziliensis isolated exclusively from cutaneous lesions, 39 with L. (L.) chagasi isolated from different sites in the same animal, and 2 with simultaneous isolation of L. (V.) braziliensis from cutaneous lesions and L. (L.) chagasi from different sites. Isolation in culture revealed the absence of Leishmania parasites in 13 animals. The results obtained confirm the existence of mixed infections in dogs in Rio de Janeiro and indicate the need to complement the investigation of seroreactive dogs using methods for the parasitological diagnosis and identification of Leishmania species.