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Detection of Trypanosoma cruzi and Trypanosoma rangeli infection in triatomine vectors by amplification of the histone H2A/SIRE and the sno-RNA-C11 genes

Trypanosoma rangeli is non pathogenic for humans but of important medical and epidemiological interest because it shares vertebrate hosts, insect vectors, reservoirs and geographic areas with T. cruzi, the etiological agent of Chagas disease. Therefore, in this work, we set up two PCR reactions, TcH2AF/R and TrFR2, to distinguish T. cruzi from T. rangeli in mixed infections of vectors based on amplification of the histone H2A/SIRE and the small nucleolar RNA Cl1 genes, respectively. Both PCRs were able to appropriately detect all T. cruzi or T. rangeli experimentally infected-triatomines, as well as the S35/S36 PCR which amplifies the variable region of minicircle kDNA of T. cruzi. In mixed infections, whereas T. cruzi DNA was amplified in 100% of samples with TcH2AF/R and S35/S36 PCRs, T. rangeli was detected in 71% with TrF/R2 and in 6% with S35/S36. In a group of Rhodnius colombiensis collected from Coyaima (Colombia), T. cruzi was identified in 100% with both PCRs and T. rangeli in 14% with TrF/R2 and 10% with S35/S36 PCR. These results show that TcH2AF/R and TrF/R2 PCRs which are capable of recognizing all T. cruzi and T. rangeli strains and lineages could be useful for diagnosis as well as for epidemiological field studies of T. cruzi and T. rangeli vector infections.Embora o Trypanosoma rangeli não seja patogênico para o homem, sua importância médica e epidemiológica reside no fato de compartilhar vetores, reservatórios e áreas geográficas com o Trypanosoma cruzi, agente causal da Doença de Chagas. Neste estudo, para distinguir T. cruzi de T. rangeli em vetores com infecções mistas, se utilizaram duas amplificações de PCR; TcH2AF/R para o gen da histona H2A/SIRE e TrFR2, para um gen repetitivo de ARN nucleolar Cl1 (sno-RNA-Cl1). Assim como a PCR S35/S36, ambas as reações foram capazes de detectar corretamente a presença de T. cruzi ou T. rangeli em triatomíneos infectados experimentalmente. Nas infecções mistas, o ADN de T. cruzi foi amplificado em 100% das amostras quando se utilizaram TcH2AF/R e S35/S36, enquanto T. rangeli foi detectado em 71% delas com os iniciadores TrF/R2 e em 6%, com S35/S36. Adicionalmente, em um grupo de Rhodnius colombiensis coletados na região de Coyaima (Tolima), T. cruzi foi identificado em 100% com ambas PCRs e T. rangeli em 14% delas com os iniciadores TrF/R2 e em 10%, com S35/S36. Estes resultados mostram que as reações de PCR TcH2AF/R e TrF/R2, capazes de reconhecer todas as cepas e linhagens de T. cruzi e T. rangeli, podem ser úteis no diagnóstico e também nos estudos epidemiológicos do campo com vetores infectados pelo T. cruzi e T. rangeli

Seguimiento de paciente con enfermedad de Chagas y trasplante de corazón mediante las PCR S35-S36 y TcH2AF-R

Introduction. Cardiomyopathy is the most common clinical form of Chagas' disease in Colombia, and one treatment option is a heart transplant. Tracking the behavior of the Chagas' parasite, Trypanosoma cruzi, is a priority due to the risk of post-transplant reactivation of the infection.Objective. A case is presented of a patient who had suffered from dilated chagasic cardiopathy and cardiac failure, and had subsequently undergone heart transplant. The case was monitored by PCR, histopathological and echocardiographic examinations.Materials and methods. Blood samples were drawn before and after the transplant, and post-transplant endomyocardial biopsies were taken. The extracted DNA was amplified with the TcH2AF-R and S35-S36 primers. Parasitemia was examined by the microhematocrit test. In addition, histopathological studies determined the parasite presence and transplant rejection status. Echocardiograms were administered to evaluate cardiac function.Results. Of the blood samples taken 83 and 48 days pre-transplant, the latter was positive by the S35-S36 PCR test. PCR tests in blood with both primers were negative up to the second month post-transplant. However, both PCR tests were positive by the third month post-transplant. Thereupon, the patient was treated with nifurtimox. Both tests presented negative results in blood 35 days after treatment was started and remained negative thereafter at 0, 3, 10 and 12 months post-treatment. The pathology, microhematocrit, and PCR test results from biopsies were negative on all the specified dates.Conclusions. PCR tests were used as indicators of a reactivation of trypanosomid infection in the patient. After treatment administration, PCR tests became negative. The patient's clinical evolution was favorable.Introducción. La cardiomiopatía es la forma clínica más común de la enfermedad de Chagas en Colombia, siendo el trasplante una opción para su tratamiento. Debido al riesgo de reactivación de la infección posterior al trasplante, es prioritario vigilar el comportamiento del parásito.Objetivo. Presentar el caso de un paciente con cardiopatía chagásica dilatada y falla cardiaca, a quien se le practicó trasplante de corazón y se le hizo seguimiento mediante PCR, análisis histopatológicos y ecocardiográficos.Materiales y métodos. Se tomaron muestras de sangre antes de la intervención y después de ella y de biopsias endomiocárdicas posteriores al trasplante. El ADN extraído fue amplificado con los iniciadores TcH2AF-R y S35-S36. La parasitemia se examinó mediante la técnica de microhematocrito. Se practicaron estudios histopatológicos para determinar la presencia del parásito o el rechazo del trasplante y, ecocardiográficos, para evaluar la función cardiaca.Resultados. De las muestras de sangre tomadas a los 83 y 48 días previos al trasplante, la última fue positiva por la PCR S35-S36. Hasta el segundo mes después del trasplante, ambas PCR fueron negativas. Al tercer mes después del trasplante, ambas PCR fueron positivas, por lo cual se inició tratamiento con nifurtimox. Tras 35 días de haberse iniciado el tratamiento, ambas pruebas presentaron resultados negativos, al igual que las tomadas a los 0, 3, 10 y 12 meses posteriores. Los resultados de la histopatología, del microhematocrito y de las PCR de las biopsias, fueron negativos en todas las fechas.Conclusiones. Las PCR permitieron sospechar la reactivación de la infección en el paciente, se le administró el tratamiento y posterioremente las pruebas se tornaron negativas. La evolución clínica del paciente ha sido favorable

Evaluación preliminar de la prueba comercial Chagas (Trypanosoma cruzi) IgG-ELISA® en individuos colombianos

Introduction: The diagnosis of Chagas’ disease is essential to provide early treatment and improve patients’ prognosis. The discriminatory efficiency of the serological tests varies according to the disease prevalence and the test-antigen used.Objective: To evaluate the discriminatory efficiency of the commercial kit Chagas (Trypanosoma cruzi) IgG-ELISA® (Nova Tec Immunodiagnostica GmbBH) in a group of Colombian individuals, using indirect immunofluorescence antibody testing (IFAT) and enzyme immunoassay (ELISA) tests as references.Materials and methods: Seventy-eight samples from chronic chagasic patients (36 asymptomatic and 42 symptomatic) and 21 healthy controls were included. Seventeen samples from non-infected people with Chagas’ disease epidemiological risk, seven with leishmaniasis and nine with non-chagasic cardiomyopathy were also analyzed. Real time PCR was performed on four individuals whose results differed among tests.Results: Significant differences at 450 nm optical absorbance were found (p<0.0001) when the median absorbance values of healthy controls (0.143), asymptomatic (2.401) and symptomatic (2.776) chagasic patients were compared, as well as when asymptomatic and symptomatic patients (p=0.0408) and seronegative people with epidemiological risk (0.232), cardiomyopathy (0.367) or leishmaniasis (0.337) were compared with chagasic patients (p<0.0001). Finally, there were differences among healthy controls and non-infected people with epidemiological risk (p=0.0264), patients with non-chagasic cardiomyopathy (p=0.0015) and patients with leishmaniasis (p=0.002). Real-time PCR was positive in three out of four analyzed cases.Conclusions: The commercial ELISA test allowed us to discriminate the chagasic patients from the controls. A phase II study of diagnostic tests for determining field reliability of this test is required.Introducción. El diagnóstico de la enfermedad de Chagas es fundamental para brindar un tratamiento oportuno y mejorar el pronóstico del paciente. La capacidad discriminatoria de las pruebas serológicas para el diagnóstico varía de acuerdo con la prevalencia de la enfermedad y el antígeno utilizado en la prueba.Objetivo. Evaluar la capacidad discriminatoria de la prueba comercial Chagas (Trypanosoma cruzi) IgG-ELISA® (NovaTec Immunodiagnostica GmbH) en un grupo de individuos colombianos utilizando la prueba de inmunofluorescencia indirecta (IFI) y el ensayo de inmunoabsorción enzimática (ELISA) como referencia.Materiales y métodos. Se incluyeron 78 muestras de pacientes crónicos (36 asintomáticos y 42 sintomáticos) y 21 de controles sanos. También se analizaron 17 individuos no infectados con riesgo epidemiológico para la enfermedad de Chagas, siete con leishmaniasis y nueve con enfermedad cardiaca. Se evaluaron por PCR en tiempo real cuatro individuos cuyos resultados variaron entre pruebas.Resultados. Se encontraron diferencias significativas auna densidad óptica de 450 nm (p<0,0001) al comparar la mediana de la absorbancia entre los controles sanos (0,143) y los asintomáticos (2,401) o sintomáticos (2,776), entre los asintomáticos y sintomáticos (p=0,0408), entre los seronegativos con riesgo (0,232), individuos con enfermedades cardiacas (0,367) o con leishmaniasis (0,337) y los pacientes con enfermedad de Chagas (p<0,0001), y entre los controles sanos y los pacientes seronegativos con riesgo (p=0,0264), con enfermedades cardiacas (p=0,0015) o con leishmaniasis (p=0,002). La PCR en tiempo real fue positiva en tres de los cuatro casos.Conclusiones. Esta prueba comercial de ELISA permitió discriminar a los pacientes con Chagas de los controles. Se requieren estudios de fase II para determinar las características operativas de la prueba

Characterising the KMP-11 and HSP-70 recombinant antigens' humoral immune response profile in chagasic patients

11 pages, 6 figures.-- The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2334/9/186/pre pubBackground: Antigen specificity and IgG subclass could be significant in the natural history of Chagas' disease. The relationship between the different stages of human Chagas' disease and the profiles of total IgG and its subclasses were thus analysed here; they were directed against a crude T. cruzi extract and three recombinant antigens: the T. cruzi kinetoplastid membrane protein-11 (rKMP-11), an internal fragment of the T. cruzi HSP-70 protein192-433, and the entire Trypanosoma rangeli HSP-70 protein. Methods: Seventeen Brazilian acute chagasic patients, 50 Colombian chronic chagasic patients (21 indeterminate and 29 cardiopathic patients) and 30 healthy individuals were included. Total IgG and its subtypes directed against the above-mentioned recombinant antigens were determined by ELISA tests. Results: The T. cruzi KMP-11 and T. rangeli HSP-70 recombinant proteins were able to distinguish both acute from chronic chagasic patients and infected people from healthy individuals. Specific antibodies to T. cruzi crude antigen in acute patients came from IgG3 and IgG4 subclasses whereas IgG1 and IgG3 were the prevalent isotypes in indeterminate and chronic chagasic patients. By contrast, the specific prominent antibodies in all disease stages against T. cruzi KMP-11 and T. rangeli HSP-70 recombinant antigens were the IgG1 subclass.This work was supported by Colciencias Research project No. 1203-333- 18692. IDF was supported by Colciencias and the Universidad Javeriana's Young Researcher 2008 Programme (Bogotá, Colombia). MCT and MCL were supported by P06-CTS-02242 Grant from PAI (Junta de Andalucia) and RICET-RD06/0021-0014, Spain. MS received financial support from the Brazilian agency - CNPq.Peer reviewe

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