Production of a conjugate between the rK346 antigen from Leishmania infantum and the horseradish peroxidase C for the detection of rK346 antibodies / Producción de un conjugado entre el antígeno rK346 de Leishmania infantum y la peroxidasa C de rábano picante para la detección de anticuerpos rK346

It was designed and characterized a reporter system to be captured by antibodies bound to ELISA plates. The system was designed with the rK346 from Leishmania infantum, a highly antigenic and specific protein. The rK346 was coupled to the horseradish peroxidase C (HRPc) from Armoracia rusticana using glutaraldehyde or sulfo-SMCC. Glutaraldehyde conjugation was performed in two steps. Separation of conjugates was carried out using a Sepharose S-200 in size exclusion chromatography (SEC); fractions were analyzed via HRPc activity and through ELISA plates sensitized with polyclonal anti-rK346 IgG purified from rabbit serum. A heterogeneous population of conjugates rK346-HRPc was obtained with molecular weights ranging between 109.7 ± 16.5 to 67.6 ± 10.1 kDa; with rK346-HRPc stoichiometries of 1:2; 2:1; 3:1; and 2:2. Conjugation using sulfo-SMCC was carried out first by introducing -SH groups onto the HRPc using the SATA reagent and the antigen was modified with sulfo-SMCC during 45 min. Separation and analysis of conjugates was performed similarly as with glutaraldehyde, resulting in a heterogeneous population of conjugates rK346-HRPc with molecular weights between 150.5 ± 22.6 to 80.0 ± 12.0 kDa; with rK346-HRPC stoichiometries of 2:1; 1:2; 2:2; and 1:3, with an increased conjugation efficiency in comparison with glutaraldehyde. This enables sulfo-SMCC to be used as a potential reagent for coupling the antigen to the HRPc, to design an economic, specific and easy method to apply as a reporter system, available to assess individuals at risk and/or at early and late stages of visceral leishmaniasis.

Se diseñó y caracterizó un sistema reportero para ser capturado por anticuerpos enlazados a placas de ELISA. El sistema fue diseñado con una proteína altamente antigénica y específica, la rK346 de Leishmania infantum. La rK346 fue acoplada a la peroxidasa C de rábano picante (HRPc) de Armoracia rusticana usando glutaraldehido o sulfo-SMCC. La conjugación con glutaraldehido fue realizada en dos pasos. La separación de los conjugados fue llevada a cabo a través de una cromatografía de exclusión molecular sefarosa S-200 (CES), las fracciones fueron analizadas midiendo la actividad HRPc y por placas ELISA sensibilizadas con inmunoglobulina G policlonal anti-rK346, purificada desde suero de conejo. Se obtuvo una población heterogénea de conjugados rK346-HRPc en un rango de pesos moleculares entre 109,7 ± 16,5 a 67,6 ± 10,1 kDa; con estequiometria rK346-HRPc de 1:2; 2:1; 3:1; y 2:2. La conjugación usando sulfo-SMCC se llevó a cabo primero introduciendo grupos -SH en la HRPc usando el reactivo SATA; el antígeno se modificó con sulfo-SMCC. La separación y el análisis de los conjugados se realizaron de forma similar que con el glutaraldehido, resultando en una población heterogénea de conjugados rK346-HRPc con un rango de pesos moleculares entre 150,5 ± 22,6 a 80,0 ± 12,0 kDa; con estequiometria rK346-HRPC de 2:1; 1:2; 2:2 y 1:3, y con una eficiencia de conjugación incrementada en comparación con glutaraldehido. De esta forma, se habilitó al sulfo-SMCC como un reactivo potencial para acoplar antígenos a la HRPc, como método para el diseño de un sistema reportero económico, especifico y fácil de aplicar, útil en la evaluación de individuos en riesgo y/o en estados tempranos o avanzados de leishmaniasis visceral.

Occurrences of anti-toxoplasma gondii and anti-neospora caninum antibodies in sheep from four districts of tocantins state, brazilian legal amazon region

Toxoplasmosis and neosporosis have been recognized as economically important diseases with considerable impact on the livestock industry. Little is known concerning the occurrence of Toxoplasma gondii and Neospora caninum in sheep from Tocantins state, Brazil. Here, we investigated antibodies against these parasites and associated factors in 182 sheep from Araguaína, Santa Terezinha do Tocantins, Arguianópolis and Palmeiras do Tocantins districts, Tocantins. Sheep sera were assayed for T. gondii and N. caninum IgG antibodies by indirect fluorescence antibody test (IFAT), using cut-off point at a dilution of 1:40 and 1:25 respectively. The prevalence of seropositive animal for T. gondii was 13.74% and 13.74% for N. caninum. None of the characteristics studied including reproductive problems, presence of cats, presence of dogs and veterinary care (p>0.05) was associated with occurrence of T. gondii or N. caninum infection. Only breed was identified as associated factor for the occurrence of toxoplasmosis in sheep (p<0.05). The present study is the first report on serum occurrence of T. gondii and N. caninum in sheep from the state of Tocantins, Brazil.

Toxoplasmose e Neosporose são reconhecidas por doenças economicamente importantes com impacto considerável na indústria pecuária. Pouco se sabe sobre a ocorrência de Toxoplasma gondii e Neospora caninum em ovelhas do estado do Tocantins, Brasil. Foram investigados a ocorrência de anticorpos contra estes parasitos e fatores associados em 182 ovelhas das cidades de Araguaína, Santa Terezinha do Tocantins, Arguianópolis e Palmeiras do Tocantins, Tocantins. Os soro das ovelhas foram testados para anticorpos IgG anti-T. gondii e anti-N. caninum pela Reação de Imunofluorescência Indireta (RIFI), usando os pontos de corte na diluição de 1:40 e 1:25, respectivamente. A prevalência de animais soropositivos para T. gondii foi de 13.74% e para N. caninum, 13.74%. Nenhuma das características estudadas incluindo problemas reprodutivos, presença de gatos, presença de cães e cuidados veterinários (p>0.05) foram associadas com a ocorrência infecção por T. gondii ou N. caninum. Somente raça foi identificada como fator associado à ocorrência de toxoplasmose em ovelhas (p<0.05). O presente trabalho é o primeiro relato da ocorrência sérica de T. gondii e N. caninum em ovelhas do estado do Tocantins, Brasil.

Occurrence of antibodies anti-Toxoplasma gondii and anti-Neospora caninum in dogs from Natal, RN, Brazil / Ocorrência de anticorpos contra Toxoplasma gondii e Neospora caninum em cães de Natal, RN, Brasil

The occurrence of anti-Toxoplasma gondii and Neospora caninum in dogs from the municipality of Natal, RN, Brazil, was determined. Information about the presence of these coccidia in this species was not known. Blood samples were collected from 29 domiciled dogs which inhabit areas that surround two important protected areas of Atlantic Forest (Parque da Cidade and Parque das Dunas) and another 73 dogs that were sacrificed due to Leishmania spp. infection, in Center for Control of Zoonosis (CCZ) for the municipality of Natal. It was only possible to obtain information about the gender of dogs that live in the parks area. The presence of antibodies against each parasite was determined by indirect Fluorescence Antibody Test (IFAT), with a cutoff of 16 for T. gondii and 50 for N. caninum. Of the 102 dogs examined, 13 (12.7%, 95% CI 7.0-20.8%) were T. gondii positive and three (2.9%, CI 0.6-8.4%) for N. caninum. Association between: localities of obtaining samples (parks x CCZ) and sex of animals, with the occurrence of antibodies against each of the parasites, was determined by the Fisher exact test. For T. gondii association was found with males (p = 0.027) and dogs living close to parks (p = 0.008) had higher rates of infection. Associations were not observed in relation to N. caninum.

A ocorrência de anticorpos anti-Toxoplasma gondii e anti-Neospora caninum foi determinada em cães do município de Natal, RN, onde informações sobre a presença destes coccídios nessa espécie não era conhecida. Para tanto, foram utilizados 29 cães domiciliados, que habitam áreas que circundam duas importantes Unidades de Conservação de Mata Atlântica presentes no município (Parque da Cidade e Parque das Dunas), e outros 73 cães que foram sacrificados no Centro de Controle de Zoonoses de Natal por serem positivos a Leishmania spp. Somente em relação aos cães que vivem próximos aos parques foi possível a obtenção de informações sobre o sexo dos animais. A presença de anticorpos contra cada um dos coccídios foi determinada com a Reação de Imunofluorescência Indireta com ponto de corte de 16 para T. gondii e 50 para N. caninum. Dos 102 cães examinados, 13 (12,7%, IC 95% 7,0-20,8) foram positivos para T. gondii e 3 (2,9%, IC 0,6-8,4%) para N. caninum. Associação entre: localidades de obtenção das amostras (parques x CCZ) e sexo dos animais com a ocorrência de anticorpos contra cada um dos parasitos, determinada através do teste exato de Fisher, foram positivas para T. gondii, com os machos (p = 0,027) e cães que habitam próximos aos parques (p = 0,008) apresentando maiores taxas de infecção. Associações não foram observadas em relação a N. caninum.

Malaria in pregnant women living in areas of low transmission on the southeast Brazilian Coast: molecular diagnosis and humoural immunity profile

Studies on autochthonous malaria in low-transmission areas in Brazil have acquired epidemiological relevance because they suggest continued transmission in what remains of the Atlantic Forest. In the southeastern portion of the state of São Paulo, outbreaks in the municipality of Juquitiba have been the focus of studies on the prevalence of Plasmodium, including asymptomatic cases. Data on the occurrence of the disease or the presence of antiplasmodial antibodies in pregnant women from this region have not previously been described. Although Plasmodium falciparum in pregnant women has been widely addressed in the literature, the interaction of Plasmodium vivax and Plasmodium malariae with this cohort has been poorly explored to date. We monitored the circulation of Plasmodium in pregnant women in health facilities located in Juquitiba using thick blood film and molecular protocols, as well as immunological assays, to evaluate humoural immune parameters. Through real-time and nested polymerase chain reaction, P. vivax and P. malariae were detected for the first time in pregnant women, with a positivity of 5.6%. Immunoassays revealed the presence of IgG antibodies: 44% for ELISA-Pv, 38.4% for SD-Bioline-Pv and 18.4% for indirect immunofluorescence assay-Pm. The high prevalence of antibodies showed significant exposure of this population to Plasmodium. In regions with similar profiles, testing for a malaria diagnosis might be indicated in prenatal care.

Comparative serology techniques for the diagnosis of Trypanosoma cruzi infection in a rural population from the state of Querétaro, Mexico

Immunological diagnostic methods for Trypanosoma cruzi depend specifically on the presence of antibodies and parasitological methods lack sensitivity during the chronic and “indeterminate” stages of the disease. This study performed a serological survey of 1,033 subjects from 52 rural communities in 12 of the 18 municipalities in the state of Querétaro, Mexico. We detected anti-T. cruzi antibodies using the following tests: indirect haemagglutination assay (IHA), indirect immunofluorescence assay (IFA), ELISA and recombinant ELISA (rELISA). We also performed Western blot (WB) analysis using iron superoxide dismutase (FeSOD), a detoxifying enzyme excreted by the parasite, as the antigen. Positive test results were distributed as follows: ELISA 8%, rELISA 6.2%, IFA and IHA 5.4% in both cases and FeSOD 8%. A comparative study of the five tests was undertaken. Sensitivity levels, specificity, positive and negative predictive values, concordance percentage and kappa index were considered. Living with animals, trips to other communities, gender, age, type of housing and symptomatology at the time of the survey were statistically analysed using SPSS software v.11.5. Detection of the FeSOD enzyme that was secreted by the parasite and used as an antigenic fraction in WBs showed a 100% correlation with traditional ELISA tests.

Situação epidemiológica da infecção por Toxoplasma gondii em equídeos na microrregião do Brejo Paraibano / Epidemiological situation of Toxoplasma gondii infection in equids from Brejo Paraibano microregion, Brazil

Objetivou-se com o estudo caracterizar a situação epidemiológica da infecção por Toxoplasma gondii em equídeos na microrregião do Brejo Paraibano, região Nordeste do Brasil. Anticorpos contra T. gondii foram pesquisados em 257 amostras de equídeos (204 equinos, 46 muares e sete asininos) em 26 propriedades. Para o diagnóstico sorológico utilizou-se a Reação de Imunofluorescência Indireta (RIFI) e um ponto de corte de 1:64. O número de focos encontrado foi de 46,1%. Nas amostras analisadas, a prevalência geral foi de 7,8% (I.C. 4,8-8,8). A prevalência foi de 8,3% (I.C. 4,9-13,0) para os equinos, 2,2% (I.C. 0,1-11,5) para os muares e 28,6% (I.C. 3,7-71,0) entre os asininos. Na regressão logística das variáveis observou-se que a fonte de água foi um fator de risco, pois naquelas propriedades que forneciam água corrente para os animais o risco de infecção foi 4,4 vezes maior do que naquelas propriedades que forneciam água parada (OR 4,4; I.C. 1,0-19,0). Este é o primeiro relato da presença de anticorpos contra T. gondii em equídeos nessa microrregião do estado da Paraíba. Para diminuir os riscos de infecção nestas espécies, deve-se fornecer aos animais uma água de boa qualidade, bem como evitar acesso de gatos a fontes de água e instalações onde os animais são mantidos.

The objective of the study was to characterize the epidemiological situation of Toxoplasma gondii infection in equids from Brejo Paraibano microregion, Northeastern Brazil. Antibodies against T. gondii were investigated in samples of 257 equids (204 horses, 46 mules and seven donkeys) in 26 properties. For serological diagnosis was used the Indirect Fluorescent Antibody Test (IFAT) and a cut-off of 1:64. The number of foci was found to be 46.1%. In the samples analyzed, the overall prevalence was 7.8% (C.I. 4.8-8.8). The prevalence was 8.3% (C.I. 4.9-13.0) for horses, 2.2% (C.I. 0.1-11.5) for mules and 28.6% (C.I. 3.771.0) among donkeys. Logistic regression of the variables showed that the water source was a risk factor, because in those properties that supplied running water to the animals the risk of infection was 4.4 times higher than in those properties which provided standing water (OR 4.4; C.I. 1.0-19.0). This is the first report of the presence of antibodies against T. gondii in equids in this micro-region of Paraiba State. To reduce the risk of infection in these species, good quality water should be given to the animals, as well as access of cats to water sources and facilities where animals are kept must be avoided.

Associação entre a presença de anticorpos anti-Leishmania sp. e anti-Neospora caninum em cães de Bauru, SP / Association between the presence of anti-Leishmania sp. and anti-Neospora caninum antibodies in dogs from Bauru, Brazil

The survey analyzed 100 samples of serum collected from dogs at the Center for Zoonoses Control of Bauru, randomly chosen. In the study, 65 percent of the samples were positive for leishmaniasis and 14 percent for neosporosis. The association between the presence of antibodies by the reaction of indirect immunofluorescent antibody test (IFAT) in the detection of anti-Leishmania and anti-Neospora antibodies was examined by the Fischer Exact Test (P=0.41), indicating no association between the results for Leishmania sp. and Neospora caninum (α=0.05). The absolute frequencies of the IFAT in the detection of antibodies anti-Leishmania and anti-Neospora caninum were analyzed using the Spearmann correlation coefficient for Leishmania and N. caninum titters, r=0.0975 and P=0.33, which did not indicate significant correlation between the titters for both pathogens.

The application of latent class analysis for diagnostic test validation of chronic Trypanosoma cruzi infection in blood donors

The main strategy to prevent transfusion-associated Chagas disease is the identification of T. cruzi-infected blood donors by serological screening tests, however there is no perfect serological gold standard. We evaluated an enzyme immunoassay (EIA), an indirect hemaglutination (IHA), and an indirect immunofluorescence (IIF) test for detecting T. cruzi antibodies in Brazilian blood donors. The results were submitted to latent class analysis, and a radioimmunopreciptation (RIPA) test was performed on repeatedly positive samples. Among 1951 donors, 11 (0.56) were positive by EIA, 6 (0.31) by IHA and 16 (0.82) by IIF. Six samples were positive with all tests, while 4 reacted with EIA and IIF. The RIPA was positive in 6 (75.0), 7 (66.6), and 4 (54.0) samples reacting by the EIA, IHA and IIF tests, respectively. The latent class model detected a high sensitivity rate (100) for the EIA and IIF, and a specificity rate of 99.95 and 99.69 for the EIA and IIF tests, respectively. The probability of being case according to the model was 99.92 when both EIA and IIF were positive, and 100 for the association of EIA, IIF, and IHA.

Seroprevalence of Toxoplasma gondii infection in goats by the indirect haemagglutination, immunofluorescence and immunoenzymatic tests in the region of Uberlândia, Brazil

A comparative study of the indirect haemagglutination (IHA), immunofluorescence (IFAT) and immunoenzymatic (ELISA) tests was carried out to determine the prevalence of Toxoplasma gondii antibodies in goats. One hundred seventy-four serum samples were obtained from four goat herds from the region of Uberlândia, State of Minas Gerais. The distribution of the animals, according to their origin, was as follow: 71 from herd I; 39 from herd II; 37 from herd III; and 27 from herd IV. Serum samples were analyzed by IHA, IFAT and ELISA, considering the reactivity of the serum samples at dilution ≥ 1:64 as cut off titer for the three tests. A global seroprevalence of 18.4 percent was observed, with significantly higher positivity rate in the herd II (66.7 percent) and older animals (> 36 months). A high and significant positive correlation was found between the titers obtained by the IHA versus IFAT, IHA versus ELISA, and ELISA versus IFAT. Therefore, it can be concluded that the three analyzed tests have shown to be highly concordant and appropriate for epidemiological surveys of Toxoplasma infection in goats. Although the seroprevalence of T. gondii infection in goats is relatively low in this region as compared to other regions of the country, adequate management might be useful and essential to control the infection in the goat herds

Variants of the Plasmodium vivax circumsporozoite protein (VK210 and VK247) in Colombian isolates

Phenotypic diversity has been described in the central repeated region of the circumsporozoite protein (CSP) from Plasmodium vivax. Two sequences VK210 (common) and VK247 (variant) have been found widely distributed in P. vivax isolates from several malaria endemic areas around the world. A third protein variant called P. vivax-like showing a sequence similar to the simian parasite P. simio-ovale has also been described. Here, using an immunofluorescent test and specific monoclonal antibodies, we assessed the presence of two of these protein variants (VK210 and VK247) in laboratory produced sporozoite. Both sequences were found in parasite isolates coming from different geographic regions of Colombia. Interestingly, sporozoites carrying the VK247 sequence were more frequently produced in Anopheles albimanus than sporozoites with the VK210 sequence. This difference in sporozoites production was statistically significant (p <0.05, Kruskal-Wallis); not correlation was found with parameters as the total number of parasites or gametocytes in blood from human donors used to feed mosquitoes. Previous studies in the same region have shown a higher prevalence of anti-VK210 antibodies which in theory may suggest their role in blocking the development of sporozoites carrying the CSP VK210 sequence

Detection of immunoglobulin G in the lung and liver of hamsters with visceral leishmaniasis

Several organs are affected in visceral leishmaniasis, not only those rich in mononuclear phagocytes. Hypergammaglobulinemia occurs during visceral leishmaniasis; anti-Leishmania antibodies are not primarily important for protection but might be involved in the pathogenesis of tissue lesions. The glomerulonephritis occurring in visceral leishmaniasis has been attributed to immune complex deposition but in other organs the mechanism has not been studied. In the current study we demonstrated the presence of IgG in the lung and liver of hamsters with visceral leishmaniasis. Hamsters were injected intraperitoneally with 2 x 10(7) amastigotes of Leishmania (Leishmania) chagasi and the presence of IgG in the liver and lung was evaluated at 7, 15, 30, 45, 80 and 102 days postinfection (PI) by immunohistochemistry. The parasite burden in the spleen and liver increased progressively during infection. We observed a deposit of IgG from day 7 PI that increased progressively until it reached highest intensity around 30 and 45 days PI, declining at later times. The IgG deposits outlined the sinusoids. In the lung a deposit of IgG was observed in the capillary walls that was moderate at day 7 PI, but the intensity increased remarkably at day 30 PI and declined at later times of infection. No significant C3 deposits were observed in the lung or in the liver. We conclude that IgG may participate in the pathogenesis of the inflammatory process of the lung and liver occurring in experimental visceral leishmaniasis and we discuss an alternative mechanism other than immune complex deposition

An appraissl of laboratory methods addressing roll back malaria

Os métodos de laboratório säo ferramentas importantes para se alcançar as principais metas para o controle da malária, que säo: a) Diagnóstico precoce e tratamento rápido; b) métodos preventivos, incluindo o controle do vetor; c) prevencäo e controle de epidemias e d) capacitaçäo de países endêmicos para pesquisa e controle da doença. Estes säo aplicados na prática clínica para o diagnóstico individual e para o seguimento de pacientes sob tratamento específico anti-malárico. Também säo úteis em estudos epidemiológicos, pois permitem medir infecções atuais e passadas da populaçäo, assessorar a suscetibilidade à drogas, avaliar o estado funcional da populaçäo e detectar a presença de infecçäo no mosquito. Para a realizaçäo do diagnóstico precoce na prática clínica deve-se utilizar métodos para detecçäo de plasmódios ou de seus componentes (antígenos ou DNA) em hemácias. Para estudos epidemiológicos, uma maior variedade de métodos podem ser utilizados, pois o tempo näo é crucial, e podem ser feitos em um laboratório central com equipamentos especializados e automatizados. Neste artigo, avaliamos criticamente os métodos de laboratório disponíveis para: a) Detecçäo de Plasmodium em hemácias; b) detecçäo de antígenos de Plasmodium em hemácias e soro; c) detecçäo do DNA de Plasmodium; d) detecçäo de anticorpos anti-Plasmodium; e) avaliar o estado imune e e) detecçäo de esporozoítas no mosquito.

Chagas disease: from bush to huts and houses: is it the case of the Brazilian Amazon?

Two of the major problems facing the Amazon - human migration from the other areas and uncontrolled deforestation - constitute the greatest risk for the establishment of endemic Chagas disease in this part of Brazil. At least 18 species of triatomines had been found in the Brazilian Amazon, 10 of them infected with Trypanosoma cruzi, associated with numerous wild reservoirs. With wide-range deforestation, wild animals will perforce be driven into other areas, with tendency for triatomines to become adapted to alternative food sources in peri and intradomicilies. Serological surveys and cross-sectional studies for Chagas disease, carried out in rural areas of the Rio Negro, in the Brazilian Amazon, showed a high level of seropositivity for T. cruzi antibodies. A strong correlation of seroreactivity with the contact of gatherers of piaçava fibers with wild triatomines could be evidenced.

Partial protection of mice against Trypanosoma cruzi after immunizing with the TcY 72 antigenic preparation

A 72 kDa Trypanosoma cruzi glycoprotein recognized by the 164C11 monoclonal antibody (IgM isotype) was purified by preparative electrophoresis. The antigenic preparation obtained, named TcY 72, was used to immunize C57Bl/10 mice. The following results were observed after immunization: (1) induction of higher titres of IgG than IgM antibodies, as evaluated by indirect immunofluorescence; (2) significant DTH after infection of epimastigotes in mice footpads: (3) peak parasitemia in immunized mice was significantly reduced and animals were negative by 13 days post-infection, although the mice still succumb to infection: (4) the phenotypic analysis of spleen cell populations showed a decrease in the CD4/CD8 ratio in immunized mice. Taken as a whole, these findings indicate that TcY 72 is immunogenic and potentially important for protective immunity.

Trypanosoma cruzi: amastigote polymorphism defined by monoclonal antibodies

We have raised monoclonal antibodies (mAbs) directed towards amastigote forms of Trypanosoma cruzi, and shown that mAbs 1D9 and 4B9 are carbohydrate while mAb 4B5 activity is resistant to periodate oxidation of the antigen. Here we used an ELISA to quantitate and compare the expression of surface epitopes on fixed parasites among different parasite isolates. The expression of markers varied among T. cruzi amastigotes isolated from infected cells or after extracellular differentiation of trypomastigotes. Moreover, we also observed an extensive polymorphic expression of these epitopes among amastigotes derived from different strains and clones. For instance, mAb 2C2 strongly and evenly reacted with 9 strains and clones (G, Y, CL, Tulahuen, MD, and F, and clones Sylvio X-10/4, D11, and CL.B), with absorbance at 492 nm (A492 nm) from 0.6 to 0.8. By contrast, mAb 4B5 had a higher expression in Tulahuen amastigotes (around 0.9 at 492 nm) whereas its reactivity with amastigotes from clones CL.B, Sylvio X-10/4 and D11 was much lower (around 0.4). mAb 1D9 displayed an interesting pattern of reactivity with amastigotes of the different strains and clones (A492 nm of G>D11ÝSylvio X-10/4 = MD>Tulahuen = F = Y>CL>CL.B). Finally, we observed that mAb 4B9 had the lowest reaction with the parasites studied, with higher values of A492 nm with Y strain (around 0.6) and lower values with Tulahuen, F and CL.B strains (around 0.2). Immunoblotting analysis also showed extensive variations among amastigotes of the various parasite isolates and mAbs 4B9, 1D9 and 4B5 revealed significant differences in expression between clones and parental strains. These data describe a previously uncharacterized polymorphism of T. cruzi amastigote surface components

Combinations of three immunological assays for detecting anti-Toxoplasma IgG in the sera of patients infected with Toxoplasma gondii.

Enzyme-linked immunosorbent assay (ELISA), Dot-ELISA and Dot-immunogold silver staining (Dot-IGSS) were simultaneously used to detect the specific IgG against Toxoplasma gondii in 65 patients infected with the protozoa. The positive rates were 86.51%, 92.51% and 98.64%, respectively. When ELISA and Dot-ELISA results were put together, the positive rate increased to 95.38%. When Dot-IGSS results were combined with those of ELISA or Dot-ELISA, the positive rate was raised to 100%. The difference in positive rate between ELISA and Dot-IGSS was significant (x2 = 6.93, p < 0.01), but no statistically significant differences were found between ELISA and Dot-ELISA or between Dot-ELISA and Dot-IGSS. Paired comparison of the reacting intensities of the sera in the 3 assays showed the correlations were highly significant (p < 0.001), with r = 0.608 between Dot-IGSS and Dot-ELISA, r = 0.8194 between Dot-IGSS and ELISA and r = 0.517 between Dot-ELISA and ELISA. Hence combination of different serological assays may increase their sensitivity and specificity for detecting the anti-Toxoplasma antibodies.

Métodos de triagem para malária, em doadores de sangue de área endêmica / Screening methods for malaria, in blood donors in endemic area

Relizou-se um estudo controlado para comparar a presença parasitemiaa e anticorpos antiplasmódios em doadores expostos ao risco de infecçäo malárica, segundo os critérios fixados nas Normas Técnicas em Hemoterapia, do Miníterio da Saúde. Eles estabelecem a rejeiçäo dos candidatos à doaçäo que apresentaram quadro febril há 30 dias e malária há 12 meses, ou que frequentaram área de malária há 6 meses. Foram estudados 395 candidatos incluídos nos critérios de rejeiçäo (expostos) e 383 candidatos aptos (controles), selecionados na triagem de doadores do Hemocentro de Manaus (AM). Em ambos os grupos, realizou-se a gota espessa e o teste da laranja acridina (QBC) para o diagnóstico parasitológico e a imunofluorescência indireta para a pesquisa de anticorpos IgM e IgG ao Plamodium vivax e falciparum. Observou-se diferença significante entre expostos e controles em relaçäo à presença de parasitemia, porém mäo para a presença de anticorpos antiplasmódios (P<0.05). O QBC foi mais sensível do que a gota espessa para detectar parasitemia e a concordância entre ambos foi de 0.66 ao Indice Kappa