Protective immunity against Trypanosoma cruzi provided by oral immunization with Phytomonas serpens: role of nitric oxide.

We have previously demonstrated that Phytomonas serpens, a tomato parasite, shares antigens with Trypanosoma cruzi, the protozoa that causes Chagas' disease. These antigens are recognized by human sera and induce protective immunity in Balb/c mice. In the present study, inducible nitric oxide synthase (iNOS) knockout (KO) mice and C57BL/6 mice treated with the nitric oxide inhibitor, aminoguanidine (AG, 50 mg kg(-1)) infected with T. cruzi, were used to demonstrate the role of nitric oxide (NO) to host protection against T. cruzi infection achieved by oral immunization with live P. serpens. A reduction in parasitaemia and an increase in survival were observed in C57BL/6 infected mice and previously immunized with P. serpens, when compared to non-immunized mice. iNOS (KO) mice immunized and C57BL/6 immunized and treated with AG presented parasitaemia and mortality rates comparable to those of infected and non-immunized mice. By itself, immunization with P. serpens did not induce inflammation in the myocardium, but C57BL/6 mice so immunized showed fewer amastigotes nests in the heart following an acute T. cruzi infection than those in non-immunized mice. These results suggest that protective immunity against T. cruzi infection induced by immunization with P. serpens is dependent upon enhanced NO production during the acute phase of T. cruzi infection.

Classification of trypanosomatids from fruits and seeds using morphological, biochemical and molecular markers revealed several genera among fruit isolates.

Trypanosomatids are widespread in several plant families and although most isolates have been classified as Phytomonas, other trypanosomatid genera can also infect plants. In order to assess the natural occurrence of non-Phytomonas trypanosomatids in plants we characterized 21 new trypanosomatid cultures, 18 from fruits and three from seeds of 17 plant species. The trypanosomatids from fruit and seeds were compared in terms of morphological, growth, biochemical and molecular features. The high diversity among the isolates permitted the classification of the new flagellates into the genera Crithidia and Leptomonas as well as Phytomonas. The data showed that natural fruit infection with non-Phytomonas trypanosomatids is more common than usually thought, being detected in 43% of the fruit isolates.

Veneza zonata (Hemiptera: Coreidae)/trypanosomatid relationship: action of hemolymph in vitro and experimental infection.

The defense response of Veneza zonata (Hemiptera: Coreidae) against three different trypanosomatid infections was assessed: (1) strain 714TD, a Leptomonas which has V. zonata as vector of a plant trypanosomatid, (2) strain 563TD, a Leptomonas isolated from the digestive tract of Euchistus heros (Hemiptera: Pentatomidae), and (3) Leishmania (L.) amazonensis, a human parasite that cannot infect V. zonata. Experiments with V. zonata hemolymph showed agglutination only of L. (L.) amazonensis culture forms and hemocytic recognition was more intense with this strain. L. (L.) amazonensis also activated the prophenoloxidase system, whereas strains 714TD and 563TD did not activate this system but rather seemed to inhibit phenoloxidase activity. No flagellates were seen in the digestive tract, hemolymph, or salivary glands in insects infected with L. (L.) amazonensis. The digestive tract, the hemolymph, and the salivary glands of insects fed on tomatoes inoculated with 714TD are sequentially invaded by the flagellate, which is inoculated in plants together with saliva. Insects fed on tomatoes inoculated with 563TD exhibited culture forms in the digestive tract (6 days after) and hemocoel (three additional days); however, they died 12 to 14 days after exposure. The salivary glands in insects inoculated in the hemocoel with 714TD strain are rapidly invaded, whereas those with 563TD culture forms died approximately 24 h after infection. Bacterial proliferation in the hemocoel and hemocyte surface blebbing were seen in insects infected only with 563TD strain as the probable pathogenic mechanism of insect death.

Surface component characterization as taxonomic tools for Phytomonas spp identification.

The genus Phytomonas arbitrarily includes all protozoa of the family Trypanosomatidae isolated from plants, but its differentiation is a complex task. The phase separation technique using Triton X-114 was used to analyze hydrophobic and hydrophilic surface proteins in ten strains of Phytomonas isolated from various fruits. The iodination of surface proteins by the Iodo-Gen method was also used for Phytomonas isolates from tomatoes, corn and annatto, Herpetomonas samuelpessoai and Crithidia fasciculata. The distribution of protein-bound radioactivity in acrylamide gels was determined by autoradiograms and showed the presence of protein bands of 36-68 kDa in all strains of Phytomonas: there were two major bands at 88 kDa and 94 kDa, with minor bands at 36 kDa and 142 kDa in H. samuelpessoai; and there were three bands at 74, 86 and 94 kDa, with minor bands at 23 kDa and 105 kDa in C. fasciculata. The results demonstrated that samples of plant parasites can be clearly differentiated from H. samuelpessoai and C. fasciculata. These plant parasites were also submitted to polysaccharide analysis by gas-liquid chromatography of the corresponding alditol acetate. Arabinose, galactose, glucose and mannose, were the major monosaccharides found, while fucose, rhamnose and xylose were found in smaller amounts. The results of all these methods indicated that, after extension to a wider range of trypanosomatid strains, they may be useful in Phytomonas taxonomy.

Genetic variability of trypanosomatids isolated from phytophagous hemiptera defined by morphological, biochemical, and molecular taxonomic markers.

In the present study, we investigated the genetic variability among 49 new isolates of trypanosomatids from phytophagous Hemiptera by means of morphological characters, growth features, and biochemical (enzymes of ornithine-arginine cycle) and molecular markers (based on spliced-leader, and ribosomal genes). From 402 phytophagous insects dissected and examined for the presence of trypanosomatids, 228 species belonging to Pyrrhocoridae, Coreidae, Lygaeidae, and Pentatomidae families harbored trypanosomatids in their salivary glands, or digestive tubes. Among these insects, 211 carried promastigotes and only 17 had choanomastigote forms. The results show a strong association among morphology, growth features, and biochemical and molecular markers and reveal the genetic diversity of the isolates, which were assigned to Crithidia, Phytomonas, and Leptomonas; we found genetic polymorphism within all these genera, thus indicating high genetic variability among trypanosomatids from phytophagous insects.

Evaluation of the western blot in the confirmatory serologic diagnosis of Chagas' disease.

The Western blot method, using antigens from epimastigote forms of the Trypanosoma cruzi Y strain, was evaluated for the confirmatory diagnosis of Chagas' disease. Serum samples were obtained from 136 chagasic patients (Group I), 23 patients with inconclusive serologic results for Chagas' disease (Group II), 53 patients with other diseases (Group III), and 50 healthy individuals (Group IV). The Western blot results for Group I gave a confirmatory diagnosis of Chagas' diseases in 118 (86.80%), an indeterminate pattern of reactivity in 16 (11.76%), and a negative pattern in only two (1.47%). Of the samples from Groups II, III and IV, none had a positive result in the Western blot; 60.86%, 52.83%, and 16.0%, respectively, showed indeterminate results; and 39.13%, 47,16% and 84,0%, respectively, showed negative results. The Western blot method showed a sensitivity of 86.60%, a specificity of 100.0%, a positive predictive value of 100.0%, and a negative predictive value of 73.50%, and the concordance coefficient kappa was high (0.7789). The results suggest that the previous serologic results for Chagas' disease could be confirmed by Western blot for the detection of specific antibodies to T. cruzi antigenic fractions, which may reduce the medical, legal, and social consequences of an inconclusive serologic result for Chagas' disease and also underscore the need for additional studies for continued efforts in the development of an ideal standard confirmatory test for Chagas' disease.

Toxoplasma gondii: II.Caracterizaçäo antigênica de taquizoíto de oito amostras / Toxoplasma gondii: II.Tachyzoite antigenic characterization of eigth strains

O amostras de T. gondii - LIV IV, LIV V e S 11 isoladas de suínos, RH e VPS de seres humanos, AS 28 de camundongos, HV III de cäo e CN de gato - foram analisadas com o objetivo de verificar a existência de possíveis diferenças na resposta imune quando inoculadas em coelhos. Através da técnica de ELISA, näo foram constatadas diferenças entre as oito amostras estudadas. Todas as amostras reagiram de forma semelhante com soros homólogos e heterólogos. A suspensäo antigênica, constituída de extrato celular total, mostrou-se eficiente no ELISA teste indireto, já que os soros positivos reagiram fortemente e os soros negativos näo apresentaram reaçäo contra os antígenos testados. A análise das amostras, pela técnica de Western blot, revelou que os isolados de T. gondii compartilham vários antígenos com algumas variaçöes. Dentre as bandas reconhecidas no Western blot, três foram comuns a todas as amostras: a p33 (33-37 kDa), p54 (52-55 kDa) e a p66 (66 kDa). A amostra HV III, isolada recentemente de um cäo, foi a que mais diferiu no perfil antigênico. Essa amostra näo apresentou três antígenos (50, 70 e 75 kDa) presentes nas demais amostras. Apenas dosi antígenos, um de 62 kDa da CN e outro de 67 kDa da LIV IV, foram amostra-específicos

Avaliação de métodos alternativos para o diagnóstico laboratorial confirmatório da Doença de Chagas / Evaluation of alternative methods for the confirmatory diagnosis of Chagas disease

Tendo em vista a ocorrência de resultados falso-positivos, falso-negativos, contraditórios e duvidosos em todos os métodos rotineiramente usados para o diagnóstico laboratorial da doença de Chagas (DC), o objetivo deste trabalho foi avaliar métodos alternativos que pudessem contribuir para o diagnóstico laboratorial confirmatório da DC. Entre eles, a detecção de antígenos circulantes do Trypanosoma Cruzi (T. cruzi) através de imunodifusão radial dupla (IDRD), contra-imunoeletroforese (CIE), eletroforese em gel de poliacrilamida (SDS-PAGE) e Western Blot (WB); a detecção de antígenos urinários de T. cruzi por SDS-PAGE, WB e enzimaimunoensaio (ELISA) de “sanduíche” de duplo anticorpo, e a detecção de anticorpos específicos contra frações antigênicas do T. cruzi pelo método do WB. Para tanto, utilizam-se 4 grupos de amostras de soros obtidos de 203 pacientes com DC (crônica), de 23 pacientes com resultados sorológicos prévios inconclusivos para DC, de 53 pacientes com outras doenças e de 50 indivíduos sadios. Também se utilizam 4 grupos de amostrasde urina obtidas de 50 pacientes com DC (35 crônica e 15 congênita), de 20 pacientes com DC crônica, de 11pacientes com outras doenças e de 13 indivíduos sadios. Os resultados obtidos revelaram positividade de 4,70 por cento a 25,00 por cento na IDRD e de 50,00 por cento na CIE. As técnicas de SDS-PAGE e WB não permitiram detectar a presença de antígenos circulantes de T. cruzi. Com as amostras de urina, a SDS-PAGE apresentou frações protéicas com peso molecular aparente de 20 a 84KDa; no teste de WB, a maioria dessas frações foram reconhecidas tanto pelo sorohiperimune de coelho anti-T.cruzi como pelo soro pré-imune de coelho, caracterizando inespecificidade de reconhecimento.. Através doELISA detectaram-se antígenos urinários de T.cruzi em 50,00 por cento das amostras de pacientes com DC (formas crônicas e congênita), em 45,00 por cento das amostras de pacientes com outras doenças e em nenhuma amostra de indivíduos sadios, resultando em índices de 50,00 por cento de sensibilidade e 79,20 por cento de especificidade. Das 136 amostras de soros de pacientes com DC, 118 (86,80 por cento) tiveram o resultado sorológico prévio confirmado pelo teste de WB, 16 (11,76 por cento) padrão indeterminado e 2 (1,47 por cento) padrão negativo; das 23 amostras com resultados sorológicos prévios inconclusivos, nenhum apresentou WB positivo, sendo 14 (60,86 por cento) com resultados indeterminado e 9 (39,13 por cento), negativo. Das 53 amostras de soros de pacientes com outras doenças, 28 (52,83 por cento) apresentaram resultado indeterminado e 25 (47,16 por cento), negativo no WB e das 50 amostras de soros de indivíduos sadios, apenas 8 (16,00 por cento) mostraram reatividade com padrão indeterminado e 42 (84,00 por cento) padrão negativo no WB. Esses valores corresponderam a índices de 86,80 por cento de sensibilidade e 100 por cento de especificidade. Os resultados obtidos permitem sugerir que os resultados sorológicos prévios para o diagnóstico da DC podem ser confirmados por diferentes métodos, principalmente pelo ELISA, para a detecção de antíngenos urinários de T. cruzi, e pelo teste de WB, para a detecção de anticorpos específicos contra frações antigênicas do T. cruzi que poderiam contribuir para limitar as conseqüências médicas, legais e sociais de um resultado sorológico prévio inconclusivo para o diagnóstico da DC. Esses resultados reforçam a necessidade de estudos adicionais e de contínuo esforço para o desenvolvimento de teste padrão ideal para o diagnóstico laboratorial confirmatório da DC.

[Congenital ChagasDisease: epidemiology, laboratorial diagnosis, prognosis and treatment]. / Doença de Chagas congênita: epidemiologia, diagnóstico laboratorial, prognóstico e tratamento.

The authors review studies about epidemiology, clinical aspects and methods used in laboratorial diagnosis of congenital Chagas'disease, emphasizing the limitations in their specificity and sensibility, and suggest alternative methods to improve the accuracy and the quality of the laboratorial diagnosis of congenital Chagasdisease, essential to an efficient treatment.

Seropositivity for anti-trypanosoma cruzi antibodies among blood donors of the "Hospital Universitário Regional do Norte do Paraná", Londrina, Brazil.

The most frequent form of acquisition of Chagas' disease in endemic areas was the transmission through the feces of contaminated triatominae. However, special attention should be paid in urban areas to transmission by blood transfusion, justifying the compulsory screening of blood donors. Early investigations at blood banks in the town of Londrina, Brazil, demonstrated that the seroprevalence of anti-Trypanosoma cruzi antibodies among blood donors was approximately 7.0% in the fifties. Further studies demonstrated practically the same seroprevalence until the eighties. In an attempt to obtain data about the real dimension of the seropositivity for anti-Trypanosoma cruzi antibodies in the region, the authors carried out a large-scale study on 45,774 serum samples from blood donors of the Hemocentro of Hospital Univesitário Regional do Norte do Paraná (HURNP), Universidade Estadual de Londrina. The immunological tests were done at the Division of Clinical Immunology of HURNP from May 1990 to December 1994. The serum samples were studied by the indirect hemagglutination assay (IHA, using kits commercially obtained from EBRAM) and by indirect immunofluorescence (IFI, using kits from LIO SERUM) with anti-human IgG conjugate (LABORCLIN). The results demonstrated that 643 serum samples were positive in both assay corresponding to a seroprevalence of 1.4%, i.e., a significant decrease in anti-Trypanosoma cruzi antibodies in the region in comparison with the previously mentioned rates. Data correlating sex and age of seropositive blood donors are presented, as well as the possible factors that may have contributed to the results observed.

Soropositividade para Doença de Chagas em pacientes com infecção pelo vírus da imunodeficiência humana (HIV) no Hospital Universitário Regional do Norte do Paraná (HURNP), Londrina, Paraná / Soropositivity to Chagas disease in patients with human immunodeficient virus infection attended at Hospital Universitário Regional do Norte do Paraná (HURNP), Londrina, Brazil

A reativação das formas indeterminadas e crônicas da Doença de Chagas (DC) tem sido associada com leucemias, linfomas, transplantes e infecção pelo vírus da imunodeficiência humana (HIV), segundo Rocha et al; Pitella; Uip et ai. Devido à disseminação da síndrome da imunodeficiência adquirida (SIDA) na América Latina, onde o DC continua sendo uma importante endemia, há um presente e crescente risco de co-infecção com o HIV e o Trypanossoma Cruzi, afirmam Amato e Neto et al. O objetivo deste trabalho é determinar a freqüência desta associação nos pacientes atendidos no HURNP, Londrina Paraná. Os resultados das reações sorológicas para DC (HAI, IFI, E ELISA) realizados em 181 soros randomicamente selecionados de pacientes com testes anti-HIV reagentes (Elisa e Western Blot) foram analisados. Entre eles, 4 (2,20%) mostraram resultados positivos em, pelo menos, duas reações sorológicas para DC; 4 (2,20%) somente na HAI; 2 (1,10% somente na IFI e 1 (0,55%) mostrou resultados duvidosos na HAI; 2 (1,10%) somente na IFI e 1 (0,55%) mostrou resultados duvidosos na HAI E IFI. O fato de que a migração tem levado a DC a novas regiões geográficas e de que a DC tem sido crescentemente diagnosticada em populações urbanas, onde há uma alta prevalência de infecção pelo HIV, segundo Rocha et al, reforça a importância de se considerar o T.cruzi outro importante patógeno oportunista associado com a SIDA, especialmente em regiões endêmicas para o T.cruzi.

Anticorpo anti-Trypanosoma cruzi como fator de reatividade cruzada na pesquisa de anticorpos anti-DNA por imunofluorescência indireta / Anti-Trypanosoma cruzi antibodies as a factor of cross-reactivity in the research of anti-DNA antibodies by indirect immunofluorescence

Na pesquisa de anticorpos anti-DNA podem ser utilizadas várias metodologias como a imunofluorescência indireta (IFI), enzimaimunoensaio (ELISA) ou radioimunoensaio (RIE) com DNA marcado com 14C, sendo que a IFI empregando Crithçidia luciliae como substrato é a mais utilizada. Sendo este um parasita flagelado da família dos tripanosomatídeos, apresenta estruturas antigênicas comuns ao Trypanosoma cruzi. Portanto, soros de pacientes com sorologia positiva para Doença de Chagas (DC) podem apresentar uma reatividade cruzada entre estes parasitas, constituindo um fator interferente na reação de IFI para pesquisa de anticorpos anti-DNA. Com o objetivo de verficar a freqüência com que esta inferência ocorre na rotina laboratorial, analisou-se os resultados obtidos na pesquisa de anti-DNA por IFI realizadas no período de junho de 1994 a julho de 1995 no Setor de Imunologia Clínica do Hospital Universitário Regional do Norte do Paraná, Londrina-Paraná. Das 927 reações realizadas, 60 (6,47%) foram reagentes, 832 (89,75%) não reagentes e 35 (3,77%) apresentaram um padrão inespecífico de fluorescência. Suspeitando-se de reatividade cruzada, foram realizadas reações sorológicas para DC (HAI e IFI) nestas amostras, sendo que 100% apresentaram resultados reagentes para ambas as reações. Estes dados confirmam a interferência dos anticorpos anti-Trypanosoma cruzi na pesquisa de anticorpos anti-DNA por IFI e alertam para a necessidade da confirmação de resultados falsos-positivos, principalmente em regiões onde a DC é endêmica.

Possible artifacts in the radioiodination of surface proteins of trypanosomatids.

Three different methods for the radioiodination of surface proteins in trypanosomatids have been comparatively analyzed. Data interpretation in the lactoperoxidase-mediated method was impaired by autoiodination of the enzyme and its tight retention by the cells. In the enzymobeads method, iodinated enzymes were released from the beads upon solubilization of the preparation with SDS, thus mixing with iodinated proteins of the trypanosomatids. Therefore, upon separation by polyacrylamide gel electrophoresis of the cell lysates, both methods yielded autoradiographic patterns in which prominent bands did not represent labeled cell surface components. In contrast, the autoradiographic patterns obtained by use of the Iodo-Gen method were apparently free of artifacts.