Identificação de espécies de Leishmania isoladas de casos humanos em Mato Grosso do Sul por meio da reação em cadeia da polimerase

As leishmanioses são zoonoses endêmicas em Mato Grosso do Sul e têm por agentes etiológicos nessa região Leishmania (Leishmania) chagasi, Leishmania (Leishmania) amazonensis e Leishmania (Viannia) braziliensis. Como método para identificação de espécies de Leishmania, a reação em cadeia da polimerase é uma ferramenta com elevada especificidade e sensibilidade. Analisaram-se 39 isolados de Leishmania criopreservados, obtidos por meio de aspirado medular e/ou biópsia de lesão, conforme a suspeita clínica. Os isolados foram submetidos à extração de DNA e à reação em cadeia da polimerase com os iniciadores: RV1/RV2 para Leishmania (Leishmania) chagasi, a1/a2 para a identificação de Leishmania (Leishmania) amazonensis e b1/b2 para Leishmania (Viannia) braziliensis. Leishmania (Leishmania) chagasi foi a única espécie identificada em 37 casos de leishmaniose visceral. Leishmania (Leishmania) amazonensis foi identificada em dois isolados de pacientes com diagnóstico de leishmaniose tegumentar. Os resultados obtidos confirmam a possibilidade do uso dos três pares de iniciadores como uma ferramenta na caracterização de isolados de Leishmania.

Epidemiological study on leishmaniasis in an area of environmental tourism and ecotourism, State of Mato Grosso do Sul, 2006-2007

The aims of this study were to carry out a serological survey of canine leishmaniasis and identify the phlebotomine fauna in the urban area of Bonito, Mato Grosso do Sul. The serological survey was conducted on a sample of 303 dogs, by means of the indirect immunofluorescence test. Phlebotomines were captured using automated light traps. The serological survey found that 30% of the dogs were seropositive, both from the center and from all districts of the town. A total of 2,772 specimens of phlebotomines were caught and the species most found was Lutzomyia longipalpis (90.4%), which corroborated its role as the vector of for canine visceral leishmaniasis in the region. Phlebotomines of the species Bichromomyia flaviscutellata (the main vector for Leishmania (Leishmania) amazonensis) and Nyssomyia whitmani (the vector for Leishmania (Viannia) brasiliensis) were also caught. The findings indicate the need for continuous epidemiological surveillance, with attention towards diminishing the vector breeding sites and the transmission of these diseases in that region.

Fauna flebotomínica (Diptera: Psychodidae) em aldeias indígenas do Estado de Mato Grosso

O estudo foi realizado em 25 aldeias indígenas pertencentes a 13 municípios do Estado de Mato Grosso. Foram identificados 4.424 exemplares de 37 espécies do gênero Lutzomyia e uma espécie do gênero Brumptomyia. Vetores da leishmaniose tegumentar americana e da leishmaniose visceral foram abundantemente capturados e representaram 28,7% (Lutzomyia whitmani) e 23,6% (Lutzomyia longipalpis), respectivamente.

Prevalência de anticorpos antileishmania spp em cães de Garanhuns, Agreste de Pernambuco

INTRODUÇÃO: Desconhecida a realidade da leishmaniose visceral canina em Garanhuns, objetivou-se investigar a ocorrência de anticorpos antileishmania spp em cães domiciliados e semidomiciliados e os possíveis fatores de risco envolvidos. MÉTODOS: Em uma primeira etapa foram coletadas 256 amostras de sangue de cães que foram submetidas à reação de imunofluorescência indireta (RIFI) na diluição 1:40. Adicionalmente, 23 amostras positivas na RIFI foram testadas com um teste rápido imunocromatográfico. Em uma segunda etapa, novas amostras de sangue de 18 cães positivos na RIFI na primeira fase do estudo foram coletadas, retestadas pela RIFI (1:40 e 1:80) e, adicionalmente, pela reação em cadeia da polimerase para pesquisa de DNA de Leishmania infantum. Ademais, 16 dessas amostras foram retestadas pelo teste rápido imunocromatográfico. RESULTADOS: Na primeira etapa, 16% das amostras foram positivas na RIFI (1:40) e apenas três (13%) foram positivas no teste rápido imunocromatográfico. Na segunda etapa, 12 amostras foram positivas na RIFI na diluição 1:40 e sete também na diluição 1:80. Nenhuma amostra foi positiva na reação em cadeia da polimerase e no teste rápido imunocromatográfico. Sinais clínicos de leishmaniose visceral ocorreram em 4,9% dos cães positivos. Não houve diferença estatística entre idade, sexo e status clínico dos cães, porém entre seus locais de origem. CONCLUSÕES: Os cães domiciliados e semidomiciliados de Garanhuns apresentam anticorpos antileishmania spp, sendo, em sua grande maioria, assintomáticos.

Leishmania (Leishmania) infantum chagasi em canídeos silvestres mantidos em cativeiro, no Estado de Mato Grosso

INTRODUÇÃO: Leishmaniose visceral é uma zoonose que acomete diversos mamíferos tendo os canídeos domésticos como principais reservatórios em ambiente urbano. A presente nota descreve a infecção de canídeos silvestres por Leishmania (Leishmania) infantum chagasi mantidos em cativeiro no Estado de Mato Grosso, Brasil. MÉTODOS: De seis raposas (Cerdocyon thous) e um cachorro vinagre (Spheotos venaticus), foram coletadas amostras de pele, medula óssea e linfonodo para detecção e caracterização de Leishmania sp pela técnica de PCR-RFLP. RESULTADOS: Todos as animais pesquisados apresentaram-se positivos para Leishmania (L.) infantum chagasi. CONCLUSÕES: Destaca-se a importância de monitoramento adequado dos mesmos, além do maior controle desta enfermidade já que estes animais estão em ambientes de recreação pública.

Aspectos ecológicos de flebotomíneos (Diptera: Psychodidae) em área urbana do município de Ponta Porã, Estado de Mato Grosso do Sul

INTRODUÇÃO: Neste trabalho, realizou-se análise faunística de flebotomíneos e levantamento dos índices de infestações (intra e peridomicílio) na área urbana de Ponta Porã/MS, de setembro de 2005 a agosto de 2007. MÉTODOS: As coletas foram realizadas com armadilhas automáticas luminosas do tipo CDC, instaladas mensalmente durante três noites consecutivas, das 18h às 6h. RESULTADOS: Foram capturados 3.946 flebotomíneos, pertencentes a oito espécies, com amplo predomínio de Lutzomyia longipalpis, apresentando os maiores índices de frequência, constância, abundância e dominância. Do total capturado, 82,9% foram de machos e 17,1% fêmeas. A média mensal de machos capturados (136,29 ± 152,01) foi significativamente maior que o número médio de fêmeas. Embora não tenham sido constatadas diferenças significativas, verificou-se que a incidência média de flebotomíneos no peridomicílio foi maior do que no intradomicílio. Uma análise de correlação revelou que três variáveis ambientais medidas (temperatura máxima, umidade relativa e precipitação pluviométrica), correlacionaram-se positivamente de forma significativa com a abundância de flebotomíneos. CONCLUSÕES: Constitui-se motivo de alerta a predominância de L. longipalpis no município de Ponta Porã, visto que implica na possibilidade de surtos de leishmaniose visceral na área, pois essa espécie é o principal vetor da Leishmania chagasi no estado bem como em outras localidades do Brasil.

Infecção natural de Lutzomyia (Lutzomyia) longipalpis (Diptera: Psychodidae) por Leishmania infantum chagasi em flebotomíneos capturados no município de Janaúba, Estado de Minas Gerais, Brasil

INTRODUÇÃO: A leishmaniose visceral tem sido notificada em quase todos os estados do Brasil, e principalmente no norte de Minas Gerais, onde a doença é endêmica. Este estudo visou detectar a infecção natural de Lutzomyia longipalpis e identificar através da técnica de PCR/RFLP a espécie de Leishmania encontrada nos flebotomíneos do município de Janaúba. MÉTODOS: Utilizando-se armadilhas luminosas, foram capturadas 1.550 fêmeas de L. longipalpis, que agrupadas em pool de 10 exemplares foram submetidas à extração e amplificação de DNA, através das técnicas de PCR genérico e cacofonia. RESULTADOS: Dos 155 pools, seis apresentaram-se positivos para Leishmania sp., sendo a taxa de infecção do município de 3,9%. Através da PCR/RFLP determinou-se que o padrão de digestão das amostras positivas foi semelhante ao da cepa referência Leishmania chagasi (MHOM/BR/74/PP75). CONCLUSÕES: A detecção de infecção natural associada a estudos sobre a epidemiologia da LV sugere que L. longipalpis esteja envolvida na transmissão de L. infantum chagasi em Janaúba, principalmente nas áreas de intensa transmissão de LV.

Phlebotomine sandflies (Diptera: Psychodidae) in Governador Valadares, a transmission area for American tegumentary leishmaniasis in State of Minas Gerais, Brazil

INTRODUCTION: A study on the phlebotomine sandfly fauna was carried out in an endemic area for American tegumentary leishmaniasis in the municipality of Governador Valadares, in the State of Minas Gerais, Brazil. METHODS: Captures were undertaken using HP light traps in four districts, on three nights per month, for one year (from January to December 2008). Correlations between climatic factors (temperature, relative air humidity and rainfall) and the numbers of sandflies collected was observed. RESULTS: 5,413 phlebotomine specimens were caught and were identified as belonging to 12 species. Of these specimens, 2,851 (52%) were females and 2,562 (48%) were males. CONCLUSIONS: Lutzomyia intermedia predominated (29.9% of the species caught), thus suggesting that they were responsible for transmission of American tegumentary leishmaniasis, together with L. whitmani, which was also found in the area (4.3%). The presence of L. longipalpis (11.9%), the main vector for visceral leishmaniasis in Brazil, is an important finding, which makes rigorous entomological surveillance of the area necessary.

Leishmanicidal activity and cytotoxicity of compounds from two Annonacea species cultivated in Northeastern Brazil

INTRODUCTION: Visceral leishmaniasis is endemic in 88 countries, with a total of 12 million people infected and 350 million at risk. In the search for new leishmanicidal agents, alkaloids and acetogenins isolated from leaves of Annona squamosa and seeds of Annona muricata were tested against promastigote and amastigote forms of Leishmania chagasi. METHODS: Methanol-water (80:20) extracts of A. squamosa leaves and A. muricata seeds were extracted with 10% phosphoric acid and organic solvents to obtain the alkaloid and acetogenin-rich extracts. These extracts were chromatographed on a silica gel column and eluted with a mixture of several solvents in crescent order of polarity. The compounds were identified by spectroscopic analysis. The isolated compounds were tested against Leishmania chagasi, which is responsible for American visceral leishmaniasis, using the MTT test assay. The cytotoxicity assay was evaluated for all isolated compounds, and for this assay, RAW 264.7 cells were used. RESULTS: O-methylarmepavine, a benzylisoquinolinic alkaloid, and a C37 trihydroxy adjacent bistetrahydrofuran acetogenin were isolated from A. squamosa, while two acetogenins, annonacinone and corossolone, were isolated from A. muricata. Against promastigotes, the alkaloid showed an IC50 of 23.3 µg/mL, and the acetogenins showed an IC50 ranging from 25.9 to 37.6 µg/mL; in the amastigote assay, the IC50 values ranged from 13.5 to 28.7 µg/mL. The cytotoxicity assay showed results ranging from 43.5 to 79.9 µg/mL. CONCLUSIONS: These results characterize A. squamosa and A. muricata as potential sources of leishmanicidal agents. Plants from Annonaceae are rich sources of natural compounds and an important tool in the search for new leishmanicidal therapies.

Risk factors for Leishmania chagasi infection in an endemic area in Raposa, State of Maranhão, Brazil

INTRODUCTION: Infection with Leishmania chagasi is the most common clinical presentation for visceral leishmaniaisis in endemic areas. The municipality of Raposa is an endemic area in State of Maranhão, Brazil, and have had registration cases of visceral leishmaniasis disease. For this reason, a cross- sectional study was conducted to evaluate the risk factors for infection with L. chagasi detected by Montenegro skin test. METHODS: The sample comprised 96% of the inhabitants of the villages of Maresia, Pantoja, and Marisol located in the municipality of Raposa, corresponding to 1,359 subjects. Data were collected using a questionnaire. Univariate and multivariate logistic regression models were applied to evaluate the association between the variables studied and infection of L. chagasi. RESULTS: The variables associated with infection upon nonadjusted analysis were a straw roof, mud walls, floors of beaten earth, presence of sand flies inside or outside of the dwelling, and bathing outdoors. Adjusted analysis showed that the presence of sand flies inside/outside the dwelling was a risk factor, and age younger than 10 years was a protective factor against asymptomatic infection. CONCLUSIONS: The results highlight the extent to which precarious living conditions of the population strengthen the epidemiological chain of visceral leishmaniasis.

Characterization of Leishmania infantum species in dogs from the urban area of Cuiabá, State of Mato Grosso, Brazil

INTRODUCTION: Visceral leishmaniasis presents urban behavior in some Brazilian cities, with domestic dogs as the main infection source. In Cuiabá, MT, canine visceral leishmaniasis was diagnosed and characterized as recommended by the Ministry of Health. METHODS: Biological samples from suspected canine carriers were analyzed by the isoenzyme electrophoresis technique. The 6PGDH enzyme and reference strain IOC/L0566 (MHOM/BR/1975/M2903) of Leishmania (Leishmania) infantum was used as one of the controls. RESULTS: Electrophoresis analysis revealed that the canine isolates belonged to the species L. (L.) infantum. CONCLUSIONS: The authors emphasize the importance of species characterization, particularly in areas of mixed infection like Cuiabá.

Diagnosis of Leishmania (Leishmania) chagasi infection in dogs and the relationship with environmental and sanitary aspects in the municipality of Palmas, state of Tocantins, Brazil

INTRODUCTION: The aim of the present study was to identify the presence of Leishmania (Leishmania) chagasi infection in dogs in the City of Palmas, Tocantins, Brazil, using the PCR technique to list the hot spots of infected dogs in the city and associate their occurrence to significant environmental changes at capture sites. METHODS: DNA was extracted from blood of dogs, and the PCR were performed with primers RV1/RV2. After screening the population studied, the regions of the city that had the highest occurrence of canine infection were detected. These sites were visited, and ecological parameters denoting anthropogenic disturbance were evaluated. RESULTS: Some important features were listed in the regions visited, such as low urbanization, lack of public collection of sewage, limited garbage collection, vacant lots with tall vegetation, decaying organic matter, and, most importantly, the occurrence of stray dogs and poultry in homes. CONCLUSIONS: The methodology for screening the population was very efficient, especially in evaluating a large number of individuals in a short time, with a high degree of automation. The results indicate an association between the observed parameters and the occurrence of infection in dogs. The model presented in the city is ideal for studies of disease progression and expansion and for the evaluation of control measures adopted for canine VL.

Clinical and laboratory alterations in dogs naturally infected by Leishmania chagasi

INTRODUCTION: Canine visceral leishmaniasis (CVL) is a zoonotic disease with different clinical manifestations. Parasitism often occurs in bone marrow, but changes have been observed in peripheral blood and serum biochemical parameters. The aim of this study was to evaluate the hematological and biochemical parameters in dogs naturally infected by Leishmania chagasi. METHODS: Eighty-five adult dogs of both sexes and various weights and ages from the Zoonosis Control Center of Fortaleza (CCZ) were used, selected by immunofluorescence assay (IFA) and considered positive with IFA titers greater than 1:40 and by visualizing amastigotes of Leishmania chagasi in smears obtained by bone marrow aspiration. The dogs (n = 85) were grouped according to clinical signs: negative (CN = 7), subclinical (CS = 10), and clinical (CC = 68). Blood samples were collected for determination of hematological and biochemical serum values. The experimental protocol was approved by the CEUA/UECE. RESULTS: The most frequent clinical signs were cachexia (77.9%), keratitis (61.8%), and lymphadenopathy (55.9%), and 86.8% of the animals showed more than one clinical sign characteristic of CVL. In CC were observed reductions in red blood cells (63%), hematocrit (72%), and hemoglobin (62%), as well as leukocytosis (33%), neutropenia (28%), thrombocytopenia (50%), uremia (45%), hyperproteinemia (53%, p<0.05), hypergammaglobulinemia (62%, p<0.01), and hypoalbuminemia (58%). CONCLUSIONS: Animals with the clinical form of the disease demonstrate hematological and biochemical changes consistent with anemia, uremia, hyperproteinemia, and hyperglobulinemia, which present themselves as strong clinical markers of visceral leishmaniasis associated with the signs previously reported.

In-house ELISA method to analyze anti-Trypanosoma cruzi IgG reactivity for differential diagnosis and evaluation of Chagas disease morbidity

INTRODUCTION: The goal was to develop an in-house serological method with high specificity and sensitivity for diagnosis and monitoring of Chagas disease morbidity. METHODS: With this purpose, the reactivities of anti-T. cruzi IgG and subclasses were tested in successive serum dilutions of patients from Berilo municipality, Jequitinhonha Valley, Minas Gerais, Brazil. The performance of the in-house ELISA was also evaluated in samples from other relevant infectious diseases, including HIV, hepatitis C (HCV), syphilis (SYP), visceral leishmaniasis (VL), and American tegumentary leishmaniasis (ATL), and noninfected controls (NI). Further analysis was performed to evaluate the applicability of this in-house methodology for monitoring Chagas disease morbidity into three groups of patients: indeterminate (IND), cardiac (CARD), and digestive/mixed (DIG/Mix), based on their clinical status. RESULTS: The analysis of total IgG reactivity at serum dilution 1:40 was an excellent approach to Chagas disease diagnosis (100% sensitivity and specificity). The analysis of IgG subclasses showed cross-reactivity, mainly with NI, VL, and ATL, at all selected serum dilutions. Based on the data analysis, the IND group displayed higher IgG3 levels and the DIG/Mix group presented higher levels of total IgG as compared with the IND and CARD groups. CONCLUSIONS: These findings demonstrated that methodology presents promising applicability in the analysis of anti-T. cruzi IgG reactivity for the differential diagnosis and evaluation of Chagas disease morbidity.

Severe coinfection of melioidosis and dengue fever in northeastern Brazil: first case report

This report focuses on a fatality involving severe dengue fever and melioidosis in a 28-year-old truck driver residing in Pacoti in northeastern Brazil. He exhibited long-term respiratory symptoms (48 days) and went through a wide-ranging clinical investigation at three hospitals, after initial clinical diagnoses of pneumonia, visceral leishmaniasis, tuberculosis, and fungal sepsis. After death, Burkholderia pseudomallei was isolated in a culture of ascitic fluid. Dengue virus type 1 was detected by polymerase chain reaction in cerebrospinal fluid (CSF); this infection was the cause of death. This description reinforces the need to consider melioidosis among the reported differential diagnoses of community-acquired infections where both melioidosis and dengue fever are endemic.