[Using S35-S36 and TcH2AF-R primer-based PCR tests to follow-up a Chagas´ disease patient who had undergone a heart transplant]. / 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.

Seguimiento de pacientes con enfermedad de chagas y trasplante de corazón mediante las PCR S35-S36 y TcH2AF-R / Using S35-S36 and TcH2AF-R primer-based PCR tests to follow-up a Chagas’ disease patient who had undergone a heart transplant

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.

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.

Reporte del primer caso de enfermedad de Chagas transplacentaria analizado por AP-PCR en Moniquirá, Boyacá / The first case of congenital Chagas' disease analyzed by AP-PCR in Colombia

Introducción. La principal vía de transmisión de la enfermedad de Chagas es por medio de los insectos vectores de la familia Reduviidae. Sin embargo, el parásito también puede ser transmitido de madres infectadas al feto in utero. Hasta la fecha no existen informes de casos de Chagas transplacentario en Colombia. Objetivo. Presentar un caso de transmisión transplacentaria ocurrido en Moniquirá, Boyacá, Colombia, y confirmarlo con el análisis de las cepas aisladas de la madre y de su bebé mediante reacción en cadena de la polimerasa (PCR) con cebadores arbitrarios. Materiales y métodos. De los hemocultivos positivos de una madre chagásica y su hijo, se extrajo el ADN de los tripanosomas y se caracterizá la especie y grupo por PCR. El genotipo de las cepas se determinó mediante AP-PCR con los iniciadores basados en los genes de b-globina (5’-CCTCACCTTCTTTCATGGAG-3’) y del ARNr 16S (5’-ACGGGCAGTGTGTACAAGACC-3’), en reacciones diferentes. Resultados. Las cepas de Trypanosoma cruzi aisladas de los hemocultivos de la madre y de su hijo mostraron el mismo perfil de amplificación por ambas pruebas de AP-PCR, concordante con el observado en las cepas T. cruzi I utilizadas como control. En los hemocultivos procedentes del reción nacido se encontrá también T. cruzi II. Conclusiones. Éste es el primer caso de enfermedad de Chagas transplacentaria reportado en el municipio de Moniquirá, que demuestra que esta forma de transmisión ocurre en el país. La presencia de infección mixta por ambos grupos de T. cruzi en las muestras del recién nacido, sugiere infección mixta en la madre, con mayor prevalencia de T. cruzi I, al menos en el hemocultivo.

Introduction. The main route of Chagas disease transmission is through vectors of the insect family Reduviidae. However, the parasite can also be transmitted from infected mothers to their fetus in utero. Until now, no cases of congenital Chagas disease have been reported in Colombia. Objective. A congenital Chagas disease case occurred in Moniquirá County, Boyacá, Colombia. It was confirmed by comparing strains isolated from the mother and her baby using polymerase chain reaction (PCR) with arbitrary primers. Materials and methods. The parasite DNA was extracted from positive blood cultures of the aflicted mother and her son. The species confirmation and group detection were performed by PCR. The strain genotypes were determined by AP-PCR with two oligonucleotides based on the genes for the b-globin (5’-CCTCACCTTCTTTCATGGAG-3’) and 16S RrNA (5’-ACGGGCAGTGTGTACAAGACC-3’), in differente reactions. Results. The T. cruzi strains isolated from the blood cultures of the mother and her son showed the same amplification profile by the two AP-PCR tests; this corresponded with profiles of the T. cruzi I strains used as controls. However, T. cruzi II was also found in the blood culture from the newborn. Conclusions. This is the first case of Chagas disease transmission reported in Moniquirá, demonstrating that this form of transmission occurs in Colombia. The presence of both groups of T. cruzi in the newborn sample suggests mixed infection in the mother as well, with a higher prevalence of T. cruzi I, at least in the mother's blood culture.

[The first case of congenital Chagas' disease analyzed by AP-PCR in Colombia]. / Reporte del primer caso de enfermedad de Chagas transplacentaria analizado por AP-PCR en Moniquirá, Boyacá.

INTRODUCTION: The main route of Chagas disease transmission is through vectors of the insect family Reduviidae. However, the parasite can also be transmitted from infected mothers to their fetus in utero. Until now, no cases of congenital Chagas disease have been reported in Colombia. OBJECTIVE: A congenital Chagas disease case occurred in Moniquirá County, Boyacá, Colombia. It was confirmed by comparing strains isolated from the mother and her baby using polymerase chain reaction (PCR) with arbitrary primers. MATERIALS AND METHODS: The parasite DNA was extracted from positive blood cultures of the afflicted mother and her son. The species confirmation and group detection were performed by PCR. The strain genotypes were determined by AP-PCR with two oligonucleotides based on the genes for the b-globin (5'-CCTCACCTTCTTTCATGGAG-3') and 16S RrNA (5'-ACGGGCAGTGTGTACAAGACC-3'), in different reactions. RESULTS: The T. cruzi strains isolated from the blood cultures of the mother and her son showed the same amplification profile by the two AP-PCR tests; this corresponded with profiles of the T. cruzi I strains used as controls. However, T. cruzi II was also found in the blood culture from the newborn. CONCLUSIONS: This is the first case of Chagas disease transmission reported in Moniquirá, demonstrating that this form of transmission occurs in Colombia. The presence of both groups of T. cruzi in the newborn sample suggests mixed infection in the mother as well, with a higher prevalence of T. cruzi I, at least in the mother's blood culture.

Caracterización molecular de los genes histona H2A y ARNsno-Cl de Trypanosoma rangeli: aplicación en pruebas diagnósticas / Molecular characterization of histone H2A and snoRNA-Cl genes of Trypanosoma rangeli: application in diagnostic tests

La aplicación de la reacción en cadena de la polimerasa (PCR) para detectar e identificar Trypanosoma rangeli y Trypanosoma rangeli presenta a menudo dificultades de interpretación. Así, algunas pruebas generan la amplificación de bandas similares provenientes de uno de los dos parásitos, fragmentos polimórficos de un mismo parásito, o la prevalencia en la detección de T. cruzi en infecciones mixtas. En este estudio se presentan y analizan los trabajos de investigación básica realizados con el objeto de diseñar y estandarizar pruebas de PCR específicas de cada parásito. Los iniciadores TcH2AF/R se diseñaron sobre la base de la región diferencial observada entre las unidades génicas que contienen los genes h2a en estos tripanosomas. Esta pareja de iniciadores amplifican un fragmento de 234 pb específico para T. cruzi (cepas I y II). Los iniciadores TrF/R2 anillan en las regiones intergénicas del fragmento génico de 801 pb codificante para seis transcritos que forman la agrupación ARNsno-Cl en T. rangeli. Estos iniciadores amplifican un fragmento de 620 pb exclusivo de las cepas KP1(-) y KP1(+) de este parásito. La aplicación de estas PCR en vectores infectados y en pacientes con enfermedad de Chagas muestra que ambas pruebas constituyen herramientas útiles para el diagnóstico y la identificación diferencial de estos tripanosomátidos.

The application of polymerase chain reaction (PCR) to detect Trypanosoma rangeli and Trypanosoma rangeli often presents interpretation challenges. For example, some tests yield the amplification of similar bands from either parasite, polymorphic fragments of the same parasite, or present deviation towards T. cruzi in mixed infections. In this study, the basic researching needed for designing and standardizating specific PCR tests for each parasite species PCR are shown and analyzed. The TcH2AF/R primers were designed on the basis of the differential gene region observed between the histone h2a genic units of these parasites. These primers amplify a specific 234 bp fragment in T. cruzi (T. cruzi I and II strains). The TrF/R2 primers anneal to the intergenic regions of an 801 bp gene fragment encoding for six transcripts that conform the snoRNA-Cl cluster in T. rangeli. These primers amplify a fragment of 620 bp exclusively in KP1(-) and KP1(+) strains of the parasite. The application of these PCR tests in infected vectors and in chagasic patients show that both tests constitute useful tools for the diagnosis and differential identification of these Trypanosomatids. Key words: histone, RNA small nucleolar (snoRNA), polymerase chain reaction (PCR), Trypanosoma.

Evaluación de las pruebas de PCR TcH2AF-R y S35-S36 para la detección de Trypanosoma cruzi en tejido cardiaco de ratón / Evaluation of TcH2AF-R and S35-S36 primers in PCR tests for the detection of Trypanosoma cruzi in mouse cardiac tissue

Introducción. El trasplante es una opción terapéutica en la cardiomiopatía chagásica. Para la detección temprana de una posible reactivación de la infección, se propone el uso de pruebas de reacción en cadena de la polimerasa (PCR) a partir de biopsias endomiocárdicas; el modelo de ratón es una aproximación preliminar para evaluar la aplicación de éstas. Objetivo. Evaluar la aplicación de las pruebas de PCR basadas en los iniciadores TcH2AF-R y S35-S36 para la detección de T. cruzi en tejido cardiaco de ratones infectados con el parásito. Materiales y métodos. Se infectaron por vía intraperitoneal dos grupos de ratones ICR de 15 y 10 individuos con 0,3 ml de PBS que contenían 1x106 tripomastigotes de la cepa MHOM/CO/2001/D.A. (T. cruzi I) o 1x104 tripomastigotes de la cepa MHOM/BR/00/Y (T. cruzi II), respectivamente. El seguimiento de la parasitemia se realizó mediante microhematocrito y presencia de parásitos en el corazón a los 30, 60 (modelo agudo), 100 y 150 (modelo crónico) días por medio de histopatología y de las PCR TcH2AF-R y S35-S36. Resultados. La histopatología mostró alteraciones en el miocardio y presencia de amastigotes en los modelos agudo y crónico. En contraste al microhematocrito y al análisis histopatológico, la PCR S35-S36 permitió la detección de ambas cepas del parásito. La PCR TcH2AF-R detectó la cepa T. cruzi I con un desempeño superior al microhematocrito y al análisis histopatológico. Conclusiones. El uso de ambas pruebas de PCR puede ser útil en la confirmación de la reactivación de la infección postrasplante.

[Evaluation of TcH2AF-R and S35-S36 primers in PCR tests for the detection of Trypanosoma cruzi in mouse cardiac tissue]. / Evaluación de las pruebas de PCR TcH2AF-R y S35-S36 para la detección de Trypanosoma cruzi en tejido cardiaco de ratón.

INTRODUCTION: Heart transplant is a therapeutic option in the treatment of chagasic cardiomyopathy. For early detection of Chagas reactivation cases, the use of PCR tests using endomyocardial biopsies has been proposed. Development of an animal model will be the first step in evaluating the applicability of this approach. OBJECTIVE: PCR tests based on the TcH2AF-R and S35-S36 primers were evaluated for the detection of T. cruzi in heart tissue of mice experimentally infected with the parasite. MATERIALS AND METHODS: Two groups of ICR mice of 15 and 10 individuals were infected by intraperitoneal injection with 0.3 ml of PBS containing 1 x 10(6) trypomastigotes of the MHOM/CO/2001/D.A. (T. cruzi I) strain or 1 x 10(4) trypomastigotes of MHOM/BR/00/Y (T. cruzi II) strain. Parasitemia and cardiac parasitic infection were determined at 30, 60 (acute model), 100 and 150 (chronic model) days by means of histopathological examination and by PCR, using the TcH2AF-R and S35-S36 primers. RESULTS: The histopathological findings revealed alterations in the heart and the presence of intracellular amastigotes in acute and chronic models. In contrast to parasitemia levels and histopathological analyses, S35-S36 PCR detected infections in mice that were infected with either parasite strain. TcH2AF-R PCR detected T. cruzi I-infected mice earlier and more frequently than inspection for parasitemia or histopathological examination. CONCLUSIONS: Applying PCR tests with both primers proved superior for Chagas disease confirmation over currently standard detection methods.

[Acute Chagas disease in Colombia: a rarely suspected disease. Report of 10 cases presented during the 2002-2005 period]. / Enfermedad de Chagas aguda en Colombia, una entidad poco sospechada. Informe de 10 casos presentados en el periodo 2002 a 2005.

INTRODUCTION: In Colombia, reported cases of acute Chagas disease are sporadic. OBJECTIVE: Ten cases were described that had been reported to the Parasitology Laboratory of the Colombian National Health Institute between December 2002 and November 2005. MATERIALS AND METHODS: Information from clinical records, epidemiological report forms, laboratory and blood tests was collated. In addition the following data were compiled: demographic variables, clinical findings, results of laboratory tests and other exams (such as peripheral blood smears), IFAT for IgG antibodies, isolation in culture medium, inoculation in mice, polymerase chain reaction tests and isoenzyme eletrophoresis. RESULTS: All the cases presented in known endemic areas for Chagas disease transmission in Colombia. Three cases were from Putumayo Province, two each from the provinces of Arauca, Casanare, Norte de Santander and one from Santander Province. The probable mode of transmission was vector-borne. Seven cases presented in adults aged 18 to 50, three in children aged 6 months to 2 years. Seven were male and three were female. The most frequent symptom was fever in nine cases. Signs of portal of entry were rare; only one patient presented a possible Romahia's sign. Three patients presented myocarditis, two acute cardiac failure and one cardiac tamponade. Parasitemia was evident in nine cases; five had positive IgG serological tests; five cases were confirmed through parasite isolation; isoenzyme electrophoresis showed Trypanosoma cruzi group I. CONCLUSIONS: Clinical variability prevailed. In none of the cases was a clinical diagnosis suspected. The diagnosis was made and confirmed through laboratory tests alone. The results highlight the importance of including this disease in the differential diagnosis of febrile syndrome in endemic regions due to its good response to etiological treatment and thereby preventing its progression to the chronic phase.

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.

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 percent of samples with TcH2AF/R and S35/S36 PCRs, T. rangeli was detected in 71 percent with TrF/R2 and in 6 percent with S35/S36. In a group of Rhodnius colombiensis collected from Coyaima (Colombia), T. cruzi was identified in 100 percent with both PCRs and T. rangeli in 14 percent with TrF/R2 and 10 percent 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 por cento das amostras quando se utilizaram TcH2AF/R e S35/S36, enquanto T. rangeli foi detectado em 71 por cento delas com os iniciadores TrF/R2 e em 6 por cento, com S35/S36. Adicionalmente, em um grupo de Rhodnius colombiensis coletados na região de Coyaima (Tolima), T. cruzi foi identificado em 100 por cento com ambas PCRs e T. rangeli em 14 por cento delas com os iniciadores TrF/R2 e em 10 por cento, 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.