Novos iniciadores para detecção de Leishmania infantum pela reação em cadeia da polimerase

Leishmania infantum causes visceral leishmaniasis (VL) in the New World. The diagnosis of VL is confirmed by parasitological and serological tests, which are not always sensitive or specific. Our aim was to design new primers to perform a Polymerase Chain Reaction (PCR) for detecting L. infantum. Sequences of the minicircle kinetoplast DNA (kDNA) were obtained from GenBank, and the FLC2/RLC2 primers were designed. Samples of DNA from L. infantum, Leishmania amazonensis, Leishmania braziliensis, Leishmania guyanensis, Leishmania naiffi, Leishmania lainsoni, Leishmania panamensis, Leishmania major and Trypanosoma cruzi were used to standardize the PCR. PCR with FLC2/RLC2 primers amplified a fragment of 230 bp and the detection limit was 0.2 fg of L. infantum DNA. Of the parasite species assayed, only L. infantum DNA was amplified. After sequencing, the fragment was aligned to GenBank sequences, and showed (99%) homology with L. infantum. In the analysis of blood samples and lesion biopsy from a dog clinically suspected to have VL, the PCR detected DNA from L. infantum. In biopsy lesions from humans and dogs with cutaneous leishmaniasis, the PCR was negative. The PCR with FLC2/RLC2 primers showed high sensitivity and specificity, and constitutes a promising technique for the diagnosis of VL.Leishmania infantum causa leishmaniose visceral (LV) no Novo Mundo. O diagnóstico de LV é confirmado por testes parasitológicos e sorológicos, os quais nem sempre são sensíveis ou específicos. Nosso objetivo foi desenhar novos iniciadores para realizar uma Reação em Cadeia da Polimerase (PCR) para detecção de L. infantum. Sequências do DNA do minicírculo do cinetoplasto (kDNA) foram obtidos do GenBank, e os iniciadores FLC2/RLC2 foram desenhados. Amostras de DNA de L. infantum, Leishmania amazonensis, Leishmania braziliensis, Leishmania guyanensis, Leishmania naiffi, Leishmania lainsoni, Leishmania panamensis, Leishmania major e Trypanosoma cruzi foram usados para padronizar a PCR. PCR com iniciadores FLC2/RLC2 amplificou um fragmento de 230 pb e detectou 0,2 fg de DNA de L. infantum.Das espécies de parasitos analisadas, somente DNA de L. infantum foi amplificado. Após sequenciamento, o fragmento foi analisado no GenBank, que mostrou homologia com L. infantum. Em análises de amostras de sangue e lesão de cão com suspeita clínica de LV, a PCR detectou DNA de L. infantum. Em amostras de lesão de humanos e cães com leishmaniose cutânea, a PCR foi negativa. A PCR padronizada com os iniciadores FLC2/RLC2 mostrou alta sensibilidade e especificidade, sendo técnica promissora para o diagnóstico de LV

Aflatoxin M1 in the urine of non-carriers and chronic carriers of hepatitis B virus in Maringa, Brazil

A exposição às aflatoxinas (AFs) na dieta é um fator de risco para o desenvolvimento do carcinoma hepatocelular (CHC) e a exacerbação da hepatite aguda em indivíduos portadores do vírus da hepatite B (VHB). O uso de biomarcadores, como a aflatoxina M1 (AFM1) na urina, produto de biotransformação da aflatoxina B1 (AFB1), permite avaliar se a população está exposta às AFs. O objetivo do presente estudo foi investigar ocorrência de AFM1 na urina de portadores e não portadores crônicos do VHB. Foi selecionado um grupo, de forma aleatória, representado por 43 portadores do VHB atendidos em dois hospitais da cidade de Maringá, Brasil, no período de Março a Junho/2008. O grupo controle foi composto por 29 voluntários adultos saudáveis anti-HBs positivo e HBsAg negativo. A determinação de AFM1 foi realizada por meio de detecção por fluorescência em sistema de cromatografia a líquido de alta eficiência com derivação pós-coluna utilizando Kobra Cell®. Das 72 amostras analisadas, 05/29 (17,2%) foram positivas para AFM1 em indivíduos não portadores do VHB, e 16/43 (37,2%) de pacientes portadores do VHB. Este estudo demonstrou a ocorrência de AFM1 na urina dos dois grupos estudados. Entretanto, há evidências de que os portadores do VHB possuam alto risco no desenvolvimento do CHC devido ao efeito aditivo pela interação entre aflatoxinas e VHB.Exposure to aflatoxins (AFs) in the diet may favour the development of hepatocellular carcinoma (HCC) and the acute exacerbation of hepatitis in chronic hepatitis B virus (HBV) carriers. Measurement of biomarkers such as aflatoxin M1 (AFM1), a metabolite of aflatoxin B1 (AFB1), in urine allows for the assessment of populations exposed to aflatoxins. The aim of this study was to investigate the occurrence of aflatoxin M1 in the urine of HBV carrier and non-carrier patients. One group included 43 randomly selected HBV carriers treated at two hospitals in the city of Maringa, Brazil, from March to June 2008. Control group consisted of 29 healthy adult volunteers with anti-HBs positive and HBsAg negative test results. Detection of AFM1 was performed by fluorescence using high performance liquid chromatography (HPLC) and post-column derivation with the Kobra Cell®. Of the 72 samples analysed, 05/29 (17.2%) AFM1 positive samples were from HBV non-carriers, and 16/43 (37.2%) of samples were from chronic HBV carriers. This study showed AFM1 in the urine of the two surveyed population. However, there is evidence that the chronic HBV carriers have a higher risk of developing HCC due to additive interaction between AFs and HBV

Topical and Intradermal Efficacy of Photodynamic Therapy with Methylene Blue and Light-Emitting Diode in the Treatment of Cutaneous Leishmaniasis Caused by Leishmania braziliensis

 Introduction: The topical and intradermal photodynamic therapy (PDT) effect of methylene blue (MB) using light-emitting diode (LED) as light source (MB/LED-PDT) in the treatment of lesions of American cutaneous leishmaniasis (ACL) caused by Leishmania braziliensis in hamsters were investigated.Methods: Hamsters were infected in the footpad with 4×107 promastigotes of L. braziliensis and divided in 4 groups: Control group was not treated, AmB group was treated with amphotericin B, MB-Id group received intradermal MB at the edge of the lesion and MB-Tp group received MB topic. After treatment with MB, the animals were illuminated using red LEDs at the 655 nm wavelength for 1 hour. The MB/LED-PDT was carried out three times a week for 12 weeks.Results: Animals of MB-Tp group presented lesion healing with significant diminution in extent of the lesion, and reduced parasite burden compared to control group; however, no significant difference was seen compared to the AmB group. MB-Tp group also showed reconstitution of the epithelium, the formation of collagen fibers, organization in the epidermis, a little disorganization and inflammation in the dermis. MB-Id was ineffective in all parameters evaluated, and it was comparable to the control group results.Conclusion: These data show that PDT with the use of MB-Tp and LED may be an alternative for the treatment of ACL. However, additional studies are being conducted to assess the potential of MB/LED-PDT, alone or in combination with conventional therapy, for the treatment of ACL

AMERICAN CUTANEOUS LEISHMANIASIS WITH UNUSUAL CLINICAL PRESENTATION AND RESPONSE TO TREATMENT

The clinical manifestations and prognosis of cutaneous leishmaniasis (CL) can be influenced by the immune response of the patient and the species of the parasite. A case of atypical clinical presentation of CL, with development of non-characteristic lesions, poor response to therapy, and a long time to resolution is reported. Confirmatory laboratory tests included parasite detection, indirect immunofluorescence, Montenegro skin test, polymerase chain reaction, and parasite identification by multilocus enzyme electrophoresis. The parasite was identified as Leishmaniabraziliensis. The lesion was unresponsive to three complete courses of N-methylglucamine antimoniate intramuscular, and to treatment with pentamidine. The patient did not tolerate amphotericin B. The lesion finally receded after treatment with intravenous N-methylglucamine antimoniate. It is essential to ensure the accuracy of diagnosis and the appropriate treatment, which can include the use a second choice drug or a different route of administration

Luz e galinhas como fatores de atração de Nyssomyia whitmani em ambiente rural, Paraná, Brasil

OBJECTIVE: To verify the influence of traps with electric light and hens as factors that attract sandflies and compare results between capture methods. METHODS: The study was conducted in the Palmital Farm, Southern Brazil. Sandfly collections were conducted with Falcão traps and an electric aspirator, fortnightly, between 8 p.m. and 11 p.m. in the presence or absence of light and hens in peridomiciliary areas, from September 1998 to June 1999. RESULTS: A total of 43,767 specimens from eight species of sandflies were collected: Nyssomyia whitmani, N. neivai and Migonemyia migonei constituting 99.9% of the total collected, with predominance of N. whitmani. The number of this species collected inside the hen's shed in the presence of hens (21,045) was greater than in their absence (10,434). In the presence of hens, with distinct intensities of light, a larger number of N. whitmani samples were collected with 3W light. In the presence of hens and light (3W), the number of N. whitmani collected with the electric aspirator (5,141) was superior to that collected with the Falcão trap (1,675). In the absence of light, with or without the presence hens, there was no difference between the numbers of N. whitmani collected with the electric aspirator or the Falcão trap. CONCLUSIONS: Hens and electric light together attract more N. whitmani to peridomicilary areas. The number of N. whitmani collected with an electric aspirator inside a hen's shed with the presence of hens and light is greater than those collected with a Falcão trap in the same conditions.OBJETIVO: Verificar a influência de armadilhas com luz elétrica e galinhas como fatores de atração de flebotomíneos e comparar os resultados entre métodos de captura. MÉTODOS: O estudo foi realizado na Fazenda Palmital, município de Terra Boa, Paraná, Brasil. As coletas de flebotomíneos foram feitas com armadilhas de Falcão e aspirador elétrico, quinzenalmente, das 20 às 23 horas, na presença ou na ausência de luz e de galinhas, no ambiente peridomiciliar, de setembro de 1998 a junho de 1999. RESULTADOS: Coletaram-se 43.767 exemplares de oito espécies de flebotomíneos; Nyssomyia whitmani, N. neivai e Migonemyia migonei representando 99,9% do total coletado, com predomínio de N. whitmani. O número de exemplares coletados na presença (21.045) foi maior do que na ausência de galinhas (10.434). Na presença de galinhas, indiferentemente das intensidades de luz, coletou-se maior número de exemplares de N. whitmani com luz de 3W. Na presença de galinhas e luz (3W) o número de N. whitmani coletado com aspirador elétrico (5.141) foi superior ao coletado com armadilha de Falcão (1.675). Na ausência de luz não houve diferença entre o número de N. whitmani coletado com o aspirador elétrico e na armadilha de Falcão, na presença ou na ausência de galinhas. CONCLUSÕES: As galinhas e a luz elétrica juntas atraem mais N. whitmani para o ambiente peridomiciliar. O número de N. whitmani coletado com o aspirador elétrico na presença de galinhas e luz no galinheiro foi maior do que o coletado na armadilha de Falcão, na mesma condição

Screening of six medicinal plant species for antileishmanial activity

This study is aimed to investigate the in vitro antileishmanial activity of ethanolic, aqueous or dichloromethane extracts of leaves, flowers, fruits or roots, of six medicinal plant species, namely, Nectandra megapotamica, Brunfelsia uniflora, Myrcianthes pungens, Anona muricata, Hymenaea stigonocarpa and Piper corcovandesis. After isolation and analysis of chemical components by ultra-high performance liquid chromatography-high-resolution tandem mass spectrometry (UHPLC-HRMS/MS), the extracts were also tested for toxicity in J774.A1 macrophages and human erythrocytes. Phenolic acids, flavonoids, acetogenins, alkaloids and lignans were identified in these extracts. Grow inhibition of promastigotes forms of Leishmania amazonensis and Leishmania braziliensis and the cytotoxicity in J774.A1 macrophages were estimated by the XTT method. The most promising results for L. amazonensis and L. braziliensis were shown by the ethanolic extract of the fruits of Hymenaea stigonocarpa and dichloromethane extract of the roots of Piper corcovadensis, with IC50 of 160 and 150 µg mL–1, resp. Ethanolic extracts of A. muricata (leaf), B. uniflora (flower and leaf), M. pungens (fruit and leaf), N. megapotamica (leaf), and aqueous extract of H. stigonocarpa (fruit) showed IC50 > 170 µg mL–1 for L. amazonensis and > 200 µg mL–1 for L. braziliensis. The extracts exhibited low cytotoxicity towards J774.A1 macrophages with CC50 > 1000 µg mL–1 and hemolytic activity from 0 to 46.1 %

Photodynamic Therapy for the Treatment of American Tegumentary Leishmaniasis: Evaluation of Therapies Association in Experimentally Infected Mice With Leishmania (Leishmania) amazonensis

Introduction: American tegumentary leishmaniasis (ATL) is a zoonotic disease caused by protozoan parasites of the genus Leishmania that affects the skin and mucous membrane. Currently, the available drugs for the treatment are injectable, with side effects, long-term treatment regimen and there is the possibility of drug resistance. Thus, alternative therapies have been tested, including photodynamic therapy (PDT). We evaluated the efficacy of PDT on its own and associated with the prescribed ATL treatment.Methods: BALB/c mice were infected with Leishmania (Leishmania) amazonensis and divided into 6 groups: Gluc+PDT, treated with Glucantime® and photodynamic therapy (PDT) with methylene blue (MB)/red LED (light-emitting diode); Gluc, treated with Glucantime®; PDT, treated with PDT with MB/red LED; Ampho+PDT, treated with amphotericin and PDT with MB/red LED; Ampho, treated with amphotericin; and control, which were infected but not treated. Two treatment cycles were performed. After 165 days of infection, the parasite load was determined.Results: Statistical differences were not found (P > 0.05) between measures of volume and thickness of the infected footpads in the treated groups when compared with the control group. However, there was a significant reduction (P < 0.05) in the parasitic load of the popliteal lymph nodes of the Gluc+PDT, Gluc, PDT and Ampho groups when compared to the control group. In the histological analysis of the infected footpads, the Gluc+PDT group presented a smaller amount of amastigote nests and lower intensity of the mononuclear infiltrate when compared to the Gluc and PDT groups.Conclusion: The results showed that although there is no significant difference in the evaluations of footpad size (thickness and volume), there is a downward measurement tendency in the Gluc+PDT group, as it can be observed by volume data and corroborated by parasite negative load

Observations on the behavior of phlebotomines in forest and domiciliary environments in an endemic area of American cutaneous leishmaniasis in southern Brazil

De novembro de 1988 a abril de 1990 capturaram-se 75.637 flebotomíneos, com armadilhas luminosas de Falcão, na fazenda Palmital, Norte do Estado do Paraná, Brasil, resultando quinze espécies. Dos insetos capturados, 95,8% o foram nos ambientes domiciliar e peridomiciliar e os restantes 4,2%, no ambiente florestal. Da totalidade de flebotomíneos, 82,0% foram capturados numa armadilha instalada dentro de um abrigo de galinhas. Dos flebotomíneos capturados em todas as armadilhas, 93,8% eram representados pelas espécies Lutzomyia migonei, Lutzomyia whitmani, Lutzomyia pessoai, Lutzomyia fischeri e Lutzomyia intermedia, todas de relevância na epidemiologia da leishmaniose tegumentar americana. L. migonei prevaleceu numa única armadilha do peridomicílio e nas demais prevaleceu L. whitmani. As densidades mensais desses insetos, obtidas usando-se somente os resultados da armadilha instalada dentro do galinheiro, foram elevadas principalmente nos meses mais quentes e úmidos. No ambiente florestal predominou a acrodendrofilia dos flebotomíneos, pois 87,9% deles foram capturados nas armadilhas instaladas a aproximadamente 10 m do solo, havendo predomínio de L. whitmani, L. fischeri, L. migonei, L. intermedia, Brumptomyia brumpti, L. monticola e L. pessoai. A carência de informações sobre a epidemiologia da leishmaniose no Estado do Paraná indica que são necessários estudos que venham esclarecer quais são as implicações das relações flebotomíneos/animais domésticos na cadeia de transmissão de Leishmania no domicílio e peridomicílio.Between November 1988 and April 1990, 75,637 phlebotomines were captured with the use of Falcão light traps on Palmital farm in the north of Paraná State, Brazil. Fifteen species were represented. The percentage of 95.8% of the insects were captured within the domiciliary area and the remaining 4.2% in the forest environment. The percentage of 62 were captured in one single Falcão light trap installed in a henhouse, where Blancasmya migonei predominated. In all the other traps Psychodopygus whilmani was predominant. B. migonei, P. whitmani, Pintomyia pessoai, Pintomyia fischeri and Psychodopygus intermedius are highly significant vectors of American cutaneous leishmaniasis and represented 93.8% of all phlebotomines captured with 10 Falcão light traps. The monthly densities of these insects were obtained from one Falcão light trap installed in a henhouse and increased especially in the warmer and more humid months. In the forest environment the phlebotomines were captured principally in traps installed about 10 meters above the ground and P. whilmani, B. migonei, P. intermedius, Brumptomyia brumpti, Psychodopygus monticola and P. pessoai were the dominant species. The need for information about the epidemiology of American cutaneous leishmaniasis calls for further investigation with a view to clarifying the implications of the relationship between the phlebotomines and domestic animals in the Leishmania cycle of transmission in domiciliary areas

Nota sobre leishmaniose canina no noroeste do Estado do Paraná, sul do Brasil

In an American cutaneous leishmaniasis endemic area in Jussara county, Paraná State, Brazil, three dogs were found to be infected by Leishmania (Viannia) braziliensis.Em área endêmica de leishmaniose tegumentar americana no Município de Jussara, Estado do Paraná, Brasil, detectaram-se três cães domésticos infectados por Leishmania (Viannia) brasiliensis