Potencial transmisión congénita de la enfermedad de Chagas en la segunda generación de jóvenes embarazadas que asistieron a su control prenatal en el departamento de Cordillera, Paraguay

Trypanosoma cruzi es el agente causal de la enfermedad de Chagas. En Paraguay se estima que existen 165.000 personas infectadas y actualmente la principal vía de transmisión es la congénita, con una prevalencia del 5% y con alrededor de 400 niños infectados nacidos por año. El departamento de Cordillera es área endémica de la Región Oriental del país, donde se implementó el Programa de Control Prenatal de Chagas en el año 1995. El objetivo de este estudio fue inferir sobre la potencial transmisión congénita de la enfermedad de Chagas en jóvenes embarazadas de segunda generación de Cordillera que asistieron a su control prenatal en el período 2010-2012, 15 años después de la implementación del Programa de Control Prenatal. De 9.588 embarazadas; 482 fueron seropositivas (5%); 2,74% en el grupo de 12 a 19 años, 4,93% de 20 a 30 años, 8,03% de 31 a 40 años y 16,11% de 41 a 48 años. Para estimar el origen de la transmisión congénita de segunda generación se tuvo en cuenta a las 72 seropositivas de 12 a 19 años y según la base de datos de las respectivas madres 61 eran seropositivas. Con esta información se estima que la transmisión a embarazadas de segunda generación de 12 a 19 años fue congénita en un 85% indicando una transmisión congénita muy significativa en la población de estudio. Esta información ayudaría al Programa Nacional de Chagas en el fortalecimiento del control de la transmisión congénita de la enfermedad de Chagas en el país.

Trypanosoma cruzi is the causative agent of Chagas disease. In Paraguay, it is estimated that 165,000 people are infected and currently the main route of transmission is the congenital one, with a prevalence of 5% and about 400 infected children are born per year. The Department of Cordillera is an endemic area of the Eastern Region of the country, where the Chagas Prenatal Control Program was implemented in 1995. The aim of this study was to infer the potential congenital transmission of Chagas disease towards second generation pregnant women from the Department of Cordillera who attended their prenatal control in the period 2010-2012, 15 years after the implementation of the Prenatal Control Program. Of 9,588 pregnant women; 482 were seropositive (5%); 2.74% in the group aged 12 to 19 years, 4.93% aged 20 to 30 years, 8.03% aged 31 to 40 years, and 16.11% aged 41 to 48 years. To estimate the origin of second generation congenital transmission, the 72 seropositive women aged 12 to 19 years were taken into account and according to the database of their respective mothers, 61 mothers of the 72 were seropositive. With this información, it can be estimated that 85% of the transmission to second generation pregnant women aged 12 to 19 years was congenital showing a very significant congenital transmission in the study population. This information could help the National Chagas Program in strengthening the control of congenital transmission of Chagas disease in the country.

Enfermedad de Chagas: Riesgo de transmisión por especies secundarias de triatominos capturados en etapa de vigilancia entomológica en las dos regiones del Paraguay

El principal vector intradomiciliar del Trypanosoma cruzi en el Cono Sur de América Latina ha sido el Triatoma infestans. En la última década se ha declarado la interrupción de transmisión de la enfermedad por esta especie en varias áreas endémicas. El T. cruzi interactúa con triatominos silvestres y reservorios mamíferos, por ello hay un riesgo permanente de la invasión de viviendas por especies secundarias como T. sordida y nativas de focos selváticos como: T. guasayana, T. guasu, Panstrongylus geniculatus, P. megistus que deben ser vigiladas para evitar el proceso de colonización de las viviendas. El objetivo fue evaluar el potencial riesgo de transmisión de la enfermedad de Chagas por especies secundarias de triatominos capturados en etapa de vigilancia entomológica en áreas endémicas de las regiones Oriental y Occidental del país. Se aplicaron técnicas moleculares asociadas a indicadores entomológicos a un total de 759 ejemplares de 4 especies de triatominos capturados en las dos regiones. Se detectó colonización del 19% por la especie T. sordida en viviendas del Departamento de Concepción. De las especies T. guasayana, T. guasu, P. geniculatus consideradas especies del ambiente selvático, se capturó al menos 1 ejemplar en cada departamento en el intradomicilio. De 759 ejemplares analizados, se detectaron 17 con infección natural con T. cruzi (2,2%), de los cuales 2 eran de P. geniculatus y 1 de T. guasayana, ambos del intradomicilio. Estos hallazgos ponen en evidencia que existe un potencial riesgo de transmisión de T. cruzi por estas especies de triatominos.

The main intradomiciliary vector of Trypanosoma cruzi in the Southern Cone of Latin America has been Triatoma infestans. In the last decade, the decrease in transmission of the disease by this species has been declared in endemic areas. T. cruzi interacts with wild triatomines and mammalian reservoirs, therefore there is a permanent risk of invasion of dwellings by secondary species such as T. sordida and native to jungle foci such as: T. guasayana, T. guasu, Panstrongilus geniculatus, P. megistus that should be monitored to avoid the process of colonization of dwellings. The objective of the study was to evaluate the potential risk of transmission of Chagas disease by secondary triatomine species captured in the entomological surveillance stage in endemic areas of the Eastern and Western regions of the country. Molecular techniques associated with entomological indicators were applied to a total of 759 specimens of 4 species of triatomines captured in the two regions. Colonization of 19% by the species T. sordida was detected in dwellings of the department of Concepción. At least one specimen of the species T. guasayana, T. guasu, P. geniculatus considered species of the jungle environment was captured in each department in the intradomiciliary environment. Of the total number of specimens analyzed (759), 17 were detected with natural infection with T. cruzi (2.2%), of which 2 were of P. geniculatus and 1 of T. guasayana both from intradomiciliary environment. These findings show that there is a potential risk of transmission of T. cruzi by these triatomine species.

Técnicas moleculares integradas a la vigilancia entomológica de vectores de la enfermedad de Chagas: Estudio del vector secundario Triatoma sordida en la Región Oriental del Paraguay / Molecular techniques integrated to the entomological surveillance of Chagas disease vectors: Study of the secondary vector Triatoma sordida in the Eastern Region of Paraguay

Las técnicas moleculares para la detección de infección natural y fuente de alimentación en vectores secundarios de la enfermedad de Chagas cuando son aplicadas a ejemplares capturados en áreas endémicas, históricamente ocupadas por Triatoma infestans, proporcionan a las investigaciones epidemiológicas respuestas más exactas con relación a la transmisibilidad de la enfermedad. El presente estudio tiene como objetivo emplear biomarcadores moleculares para evaluar el impacto de la infestación intra y peridomicilar de Triatoma sordida en viviendas bajo vigilancia entomológica de departamentos de la Región Oriental del Paraguay en el período 2007 al 2015. Un total de 559 ejemplares de T. sordida capturados en 253, 91 y 52 viviendas de los departamentos Paraguarí, San Pedro y Cordillera, respectivamente fueron analizados. La infestación detectada fue del 24% al 48% así como una elevada colonización intradomiciliar del 5% al 36% en los tres departamentos. La detección molecular de infección natural osciló entre el 14% y 44%; y en 111 ejemplares se determinó la fuente de alimentación. El marcador molecular citocromo b permitió demostrar por vez primera un elevado porcentaje de triatominos con sangre humana como fuente de alimentación, principalmente en Cordillera con un 82% (28/34 T. sordida capturados). Estos hallazgos dejan en evidencia el avance del T. sordida en la ocupación del nicho ecológico de T. cruzi y la capacidad de esta especie secundaria como vector en la transmisión de T. cruzi en comunidades de la Región Oriental

When molecular techniques for the detection of natural infection and blood meal source in secondary vectors of Chagas disease are applied to specimens captured in endemic areas, historically occupied by Triatoma infestans, provide more accurate answers to questions about transmissibility of the illness and further contribute to the epidemiological studies. The aim of this study was to evaluate the impact of intra and peridomiciliary infestation of Triatoma sordida in households from the departments of the Eastern Region of Paraguay, under entomological surveillance during the period 2007 to 2015, by using the molecular biomarkers technology. A total of 559 specimens of T. sordida captured in 253, 91 and 52 households from Paraguarí, San Pedro and Cordillera departments, respectively, were analyzed. The infestation detected was from 24% to 48% as well as a high intradomicialiary colonization from 5% to 36% in the three departments. The molecular detection of natural infections ranged from 14% to 44% and in 111 specimens the meal source was identified. The molecular marker cytochrome b allowed to demonstrate, for the first time, high frequency of triatomines with human blood as a food source, mainly in Cordillera as it was determined in 82% (28/34) of the T. sordida captured. These findings demonstrate a progress of T. sordida into the ecological niche of T. cruzi and the abillity of this secondary species as a vector of the transmission of T. cruzi in communities from the Eastern Region of Paraguay

IL-17A, a possible biomarker for the evaluation of treatment response in Trypanosoma cruzi infected children: A 12-months follow-up study in Bolivia.

BACKGROUND: The National Program for Chagas disease was implemented in Bolivia in 2006, and it greatly decreased the number of infections through vector control. Subsequently, a treatment regimen of benznidazole (BNZ) was started in seropositive school-age children living in certified vector control areas. METHODS AND FINDINGS: We conducted a 12-month follow-up study and seven blood samples were taken during and after the treatment. Serology, conventional diagnostic PCR (cPCR) and quantitative Real-time PCR (qPCR) were performed. Plasma Th1/Th2/Th17 cytokines levels were also determined. Approximately 73 of 103 seropositive children complied with BNZ, with three interruptions due to side effects. To evaluate each individual's treatment efficacy, the cPCR and qPCR values during the final 6 months of the follow-up period were observed. Among 57 children who completed follow-up, 6 individuals (11%) showed both cPCR(+) and qPCR(+) (non reactive), 24 (42%) cPCR(-) but qPCR(+) (ambiguous) and 27 (47%) cPCR(-) and qPCR(-) (reactive). Within 14 Th1/Th2/Th17 cytokines, IL-17A showed significantly higher levels in seropositive children before the treatment compared to age-matched seronegative children and significantly decreased to the normal level one-year after. Moreover, throughout the follow-up study, IL-17A levels were positively co-related to parasite counts detected by qPCR. At the 12 months' time point, IL-17A levels of non-reactive subjects were significantly higher than either those of reactive or ambiguous subjects suggesting that IL-17A might be useful to determine the reactivity to BNZ treatment. CONCLUSIONS: Plasma levels of IL-17A might be a bio-marker for detecting persistent infection of T. cruzi and its chronic inflammation.

Fuente de alimentación de ejemplares de Triatoma sordida en un área con alto riesgo de domiciliación en el Chaco Paraguayo / Blood meal source of specimens of Triatoma sordida in an area with high risk of domiciliation in the Paraguayan Chaco

En el presente estudio evaluamos indicadores entomológicos (índices de infección, colonización e infección natural) de ejemplares de Triatoma sordida capturados en el intra y peridomicilio de viviendas del Chaco Paraguayo en el período 2014 al 2016. El objetivo fue comparar con los hallazgos previamente reportados en el periodo 2010 al 2013 en la misma región. Además se ha incorporado la detección de fuente de alimentación como complemento para definir el riesgo de domiciliación de este vector secundario. Se aplicaron técnicas moleculares asociados a indicadores entomológicos y epidemiológicos a 220 ejemplares de T. sordida capturados en 67 viviendas de 24 localidades del Chaco. Se detectó infestación y colonización por T. sordida en el intradomicilio 13/67 (19%) y 5/13 (38%) y en el peridomicilio 54/67 (81%) y 43/54 (80%), respectivamente. Se detectó infección con T. cruzi en un 17,3% de los ejemplares analizados. La fuente de alimentación pudo detectarse en tan solo 13 de 220 ejemplares (6%), todos resultaron positivos para sangre de gallina y correspondían a captura en el peridomicilio. El índice de infestación intradomiciliar fue del 19%, superior al de años anteriores y similar a las zonas históricamente endémicas de la Región Occidental (18%-20%). El riesgo de transmisión intradomiciliar sigue siendo elevado porque en 3 de 5 viviendas con ninfas de T. sordida, los ejemplares estaban infectados con T. cruzi. Los indicadores entomológicos obtenidos en el presente estudio concuerdan con resultados previos de nuestro equipo, y confirman la capacidad vectorial de este triatomino secundario en la transmisión de la enfermedad de Chagas(AU)

Chagas disease: national survey of seroprevalence in children under five years of age conducted in 2008

BACKGROUND Since the early 1990s, programs to control Chagas disease in South America have focused on eradicating domiciliary Triatoma infestans, the main vector. Seroprevalence studies of the chagasic infection are included as part of the vector control programs; they are essential to assess the impact of vector control measures and to monitor the prevention of vector transmission. OBJECTIVE To assess the interruption of domiciliary vector transmission of Chagas disease by T. infestans in Paraguay by evaluating the current state of transmission in rural areas. METHODS A survey of seroprevalence of Chagas disease was carried out in a representative sample group of Paraguayans aged one to five years living in rural areas of Paraguay in 2008. Blood samples collected on filter paper from 12,776 children were tested using an enzyme-linked immunosorbent assay. Children whose serology was positive or undetermined (n = 41) were recalled to donate a whole blood sample for retesting. Their homes were inspected for current triatomine infestation. Blood samples from their respective mothers were also collected and tested to check possible transmission of the disease by a congenital route. FINDINGS A seroprevalence rate of 0.24% for Trypanosoma cruzi infection was detected in children under five years of age among the country’s rural population. Our findings indicate that T. cruzi was transmitted to these children vertically. The total number of infected children, aged one to five years living in these departments, was estimated at 1,691 cases with an annual incidence of congenital transmission of 338 cases per year. MAIN CONCLUSION We determined the impact of vector control in the transmission of T. cruzi, following uninterrupted vector control measures employed since 1999 in contiguous T. infestans-endemic areas of Paraguay, and this allowed us to estimate the degree of risk of congenital transmission in the country.

Chagas disease: national survey of seroprevalence in children under five years of age conducted in 2008.

BACKGROUND: Since the early 1990s, programs to control Chagas disease in South America have focused on eradicating domiciliary Triatoma infestans, the main vector. Seroprevalence studies of the chagasic infection are included as part of the vector control programs; they are essential to assess the impact of vector control measures and to monitor the prevention of vector transmission. OBJECTIVE: To assess the interruption of domiciliary vector transmission of Chagas disease by T. infestans in Paraguay by evaluating the current state of transmission in rural areas. METHODS: A survey of seroprevalence of Chagas disease was carried out in a representative sample group of Paraguayans aged one to five years living in rural areas of Paraguay in 2008. Blood samples collected on filter paper from 12,776 children were tested using an enzyme-linked immunosorbent assay. Children whose serology was positive or undetermined (n = 41) were recalled to donate a whole blood sample for retesting. Their homes were inspected for current triatomine infestation. Blood samples from their respective mothers were also collected and tested to check possible transmission of the disease by a congenital route. FINDINGS: A seroprevalence rate of 0.24% for Trypanosoma cruzi infection was detected in children under five years of age among the country's rural population. Our findings indicate that T. cruzi was transmitted to these children vertically. The total number of infected children, aged one to five years living in these departments, was estimated at 1,691 cases with an annual incidence of congenital transmission of 338 cases per year. MAIN CONCLUSION: We determined the impact of vector control in the transmission of T. cruzi, following uninterrupted vector control measures employed since 1999 in contiguous T. infestans-endemic areas of Paraguay, and this allowed us to estimate the degree of risk of congenital transmission in the country.

Triatoma sordida: indicadores de adaptación y transmisión de trypanosoma cruzi en intradomicilio del Chaco Paraguayo / Triatoma sordida: indicators of adaptive capacity and trypanosoma cruzi intradomicile transmission in the Paraguayan Chaco

El gran Chaco es una eco-región que incluye Argentina, Bolivia y Paraguay; donde la trasmisión vectorial de la enfermedad de Chagas por Triatoma infestans (vector principal), constituye hasta la fecha un problema de salud pública. El Chaco paraguayo ocupa el 25% de esta región, caracterizada por una baja densidad poblacional y localidades dispersas. El objetivo de este estudio fue determinar el rol potencial de la especie Triatoma sordida (vector secundario) en el ciclo doméstico de transmisión de Trypanosoma cruzi. Se aplicaron técnicas moleculares asociadas a indicadores entomológicos y epidemiológicosa 436 ejemplares de T. sordida capturados en 147 viviendas del Chaco Paraguayo. Se detectó infestación y colonización en el intradomicilio y peridomicilio por T. sórdida en 12 (8.2%) y 79 (53.7%) viviendas de las 147 evaluadas, respectivamente. Al menos un ejemplar infectado con T. cruzi fue detectado por PCR en las 12 viviendas con colonización intradomiciliar y en dos de ellas por caracterización molecular se detectó en ninfas el genotipo TC2. Adultos y ninfas en el peridomicilio de 4 viviendas dieron positivo para el genotipo TC1. Se estima un elevado riesgo de transmisión de T. cruzi intradomiciliar del 87%. Estos resultados evidencian capacidad adaptativa de esta especie en el domicilio, y un incremento de su potencial vectorial para transmitir la enfermedad de Chagas en el Chaco Paraguayo.

The Gran Chaco is an eco-region that includes Argentina, Bolivia and Paraguay; where the vectorial transmission of Chagas disease by Triatoma infestans (main vector) is a public health problem up to date. The Paraguayan Chaco occupies 25% of this region, characterized by low population density and dispersed locations. The aim of this study wasto determine the potential role of the specie Triatoma sordida (secondary vector) in the domestic cycle of transmission of Trypanosoma cruzi. Molecular techniques associated with entomological and epidemiological indicators were applied in 436 specimens of T. sordida captured in 147 dwellings of the Paraguayan Chaco. Infestation rate and colonization by T. Sordida was detected in the intradomicile and peridomiciliary in 12 (8.2%) and 79 (53.7%)of the 147 dwellings, respectively. In 12 houses were colonization was observed, at leastone specimen was PCR-T. cruzi positive, and in two of them genotype TC2 of T. cruzi were detected innymphs by molecular characterization. Adults and nymphs in the peridomiciliary of 4 houses were positive for genotype TC1.A high risk of indoor transmission of T. cruzi isestimated in 87%. These results demonstrate adaptive capacity of this specie in the intradomicile, and increased vector potential to transmit Chagas disease in the Paraguayan Chaco.

Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients.

An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease.

Diagnosis of congenital Trypanosoma cruzi infection: A serologic test using Shed Acute Phase Antigen (SAPA) in mother-child binomial samples.

Chagas congenital infection is an important health problem in endemic and non-endemic areas in which Trypanosoma cruzi-infected women can transmit the parasite to their offspring. In this study, we evaluated the antibody levels against the T. cruzi Shed Acute Phase Antigen (SAPA) in 91 binomial samples of seropositive pregnant women and their infected and non-infected children by ELISA. In 70 children without congenital T. cruzi transmission, the titers of anti-SAPA antibodies were lower than those of their seropositive mothers. In contrast, 90.5% of 21 congenitally infected children, at around 1 month of age, showed higher anti-SAPA antibody levels than their mothers. Subtracting the SAPA-ELISA mother OD value to the SAPA-ELISA child OD allowed efficient detection of most T. cruzi congenitally infected children immediately after birth, when total anti-parasite antibodies transferred during pregnancy are still present in all children born to seropositive women. A positive correlation was observed between parasitemia levels in mothers and infants evaluated by quantitative DNA amplification and anti-SAPA antibody titers by ELISA. As SAPA serology has proved to be very efficient to detect T. cruzi infection in mother-child binomial samples, it could be of extreme help for early diagnosis of newborns, in maternities and hospitals where DNA amplification is not available. This prompt diagnosis may prevent drop out of the long-term follow-up for future diagnosis and may ensure early trypanocidal treatment, which has proved to be efficient to cure infants with congenital Chagas disease.

Estandarización de la técnica PCR-RFLP del gen mitocondrial cyt b como herramienta para la identificación de fuentes de alimentación de insectos hematófagos / Standardization of a PCR-RFLP technique based on RESUMEN mitochondrial cyt b gen as a tool to identify feeding sources of hematophagous insects

La identificación de la fuente de alimentación de insectos hematófagos puede proporcionar información sobre la capacidad vectorial, patrones de alimentación en condiciones naturales y proveer indirectamente datos sobre probables reservorios de enfermedades. Varias técnicas de identificación son empleadas, entre ellas las más utilizadas son las basadas en reacciones antígeno-anticuerpo. Actualmente, se han desarrollado ensayos moleculares, algunos de ellos permiten detectar e identificar solo sangre humana. Otros como la reacción en cadena de la polimerasa (PCR) del gen mitocondrial citocromo b (cyt b), que ha mostrado alto grado de sensibilidad y especificidad, permite detectar e identificar otras especies de vertebrados. El objetivo del trabajo fue estandarizar la técnica PCR-RFLP del gen mitocondrial citocromo b (cyt b) para determinar la fuente de alimentación sanguínea de insectos. Inicialmente se realizó un análisis bioinformático para la búsqueda y alineamiento de secuencias del gen cyt b de los potenciales huéspedes, con secuencias que están disponible en el GenBank. Se utilizaron 10 muestras de sangre de potenciales huéspedes vertebrados (humano, perro, gallina y roedor) y para la reacción de PCR se empleó un par de cebadores universales que amplifican una región del gen cyt b, seguido de cortes con dos enzimas de restricción (RFLP) (Hae III y Mwo I), generando patrones de electroforesis específicos para los diferentes vertebrados. Se logró la estandarización de la técnica de PCR del gen cyt b que fue capaz de detectar ADN de al menos 1 μL de sangre observándose el producto de amplificación de 358 pb. El análisis de los patrones de bandas obtenidos con el corte de las enzimas mostró los tamaños de fragmentos esperados para humano, gallina, perro y roedor. Estos resultados muestran la utilidad de la técnica PCR-RFLP del gen cyt b que, con un simple par de cebadores seguido del corte con dos enzimas de restricción...

Identification of feeding sources of hematophagous insects can provide informationabout the vectorial capacity,feeding patternsin natural conditionsand indirectlyprovidedataon possible disease reservoirs preferences.Several identificationtechniquesare used,most of them based on antigen-antibody reactions.Recently molecular assayshave been developedand, some ofthese assayscandetect andidentificateonlyhuman blood. Otherassays,likethepolymerase chain reaction (PCR)basedonmitochondrialcytochromebgene,have shown high sensitivity and specificityallowingdetectionand identificationofothervertebrate species. The aim of this studywas tostandardizea PCR-RFLP basedonmitochondrialcytochrome bgene(cyt b) inorder to determineblood mealfrom insects.Initially bioinformatic analysiswasperformed forsearching andalignmentofcyt bsequencesofpotential hosts availableattheGenBankdatabase.Blood samplesfrom potentialvertebrate hostswere usedand PCR was performed using specificprimersthat amplify aregionofcyt bgene. Theproducts were digestedwithrestriction enzymes (RFLP),generating specificelectrophoresis patterns forseveral vertebrates. The PCR technique forcyt bgene wasstandardized allowing the detection of at least 1μLof blood.The 359 bp band wascorrectly amplified and the profiles obtained after the enzyme digestion withHaeIIIandMwoI were the expected for human, chicken, dog and rodents. These resultsshowed the usefulness of the PCR-RFLP ofcyt bgenethat,with a single pair of primersfollowed digestionusing two restriction enzymes,allowed the differentiationof thevertebrate species of our interestthrough the patterns obtained without sequencinghavingalso the advantage of detecting small volumesof blood sample.

DNA methylation-mediated silencing of PU.1 in leukemia cells resistant to cell differentiation.

In mice, the proviral integration of the Friend Spleen Focus Forming Virus (SFFV) within the PU.1 locus of erythroid precursors results in the development of erythroleukemia. SFFV integrates several kilobases upstream of the PU.1 transcription initiation start site leading to the constitutive activation of the gene which in turn results in a block of erythroid differentiation. In this study we have mapped and sequenced the exact location of the retroviral integration site. We have shown that SFFV integrates downstream of a previously described upstream regulatory element (URE), precisely 2,976 bp downstream of the URE-distal element. We have also found that SFFV persists integrated within the same location in resistant cell lines that have lost their differentiation capacity and in which case PU.1 remains silent. We have examined the methylation status of PU.1 and found that in resistant cells the nearby CpG islands remained methylated in contrast to a non-methylated status of the parental cell lines. Treatment with 5-aza-2'-deoxycytidine caused resistant cells to differentiate yet only when combined with HMBA. Altogether these results strongly suggest that methylation plays a crucial role with regard to PU.1 silencing. However, although demethylation is required, it is not sufficient to overcome the differentiation impasse. We have also showed that activation blockage of the Epo/Epo-R pathway remains despite of the absence of PU.1.

Identificación molecular de linajes y sub-linajes de Trypanosoma cruzi en niños infectados congénitamente provenientes de áreas endémicas del Paraguay / Molecular identification of Trypanosoma cruzi lineages and sublineages in congenitally infected children from endemic areas of Paraguay

La tripanosomiasis americana es una infección causada por un protozoario flagelar Trypanosoma cruzi que está diseminado por todo el continente Americano. T. cruzi es una especie heterogénea que comprende varias sub-poblaciones que circulan en hospederos vertebrados domésticos y salvajes, e invertebrados. Esta diversidad genética ha sido detectada con técnicas moleculares demostrándose que circulan clones mayoritarios en varios hospederos y en diferentes áreas geográficas de América Latina. Aislados de T. cruzi han sido divididos en dos grupos genéticos nombrados por consenso internacional T. cruzi I (linaje 2-asociado al ciclo enzoótico) y T. cruzi II (linaje 1-asociado al ciclo doméstico). La interacción de los clones infectivos de T. cruzi y el hospedero humano podrían determinar la morbilidad de la enfermedad. En este trabajo se empleó la técnica PCR para amplificar genes constitutivos de T. cruzi, como el gen 24S Ribosomal y el gen mini exón (ME) y se detectaron linajes de T. cruzi en 112 muestras (83%) de un total de 135 muestras de niños infectados provenientes de dos departamentos endémicos para la enfermedad de Chagas en Paraguay (Cordillera y Paraguarí). En cuanto a la procedencia de las mismas, 53 muestras eran de Paraguarí y 59 muestras de Cordillera. Los sub-linajes encontrados fueron: Paraguarí: 34TcIIc, 17 TcIId, 1 muestra TcIIb-IIe y 1 muestra ND (no determinada), En Cordillera: 37 TcIIc, 19 TcIId, 1 muestra TcIIb-IIe y 2 muestras no determinadas. No se encontró linaje I, selvático en ninguna de las 2 áreas estudiadas

Estrategias de afrontamiento en adolescentes / Coping strategies in adolescents

Este estudio tuvo como objetivo describir y analizar las estrategias de afrontamiento que presentan los adolescentes escolarizados. La muestra total estuvo conformada por 323 adolecentes, estudiantes (184 hombres y 139 mujeres) de Octavo Básico a Cuarto Año Medio pertenecientes a diferentes tipos Establecimientos Educacionales de la ciudad de Arica (Chile): Privado (90), Particular Subvencionado (114) y Municipal (119). A todos ellos se les aplicó la escala ASC de Frydenberg y Lewis (1997). Se encontraron diferencias significativas entre los tipos de Establecimiento Educacional, siendo el Establecimiento Particular Subvencionado el que evidenció mayor variedad de estrategias de afrontamiento utilizadas. Se discuten las implicancias a la luz de los resultados hallados.

This study aimed to describe and analyze the coping strategies that adolescents have in school. The total sample consisted of 323 adolescents, students (184 men and 139 women) from Eighth to Fourth Year Basic Middle belonging to different Educational Institutions in the city of Arica (Chile): Private (90), Private Subsidized (114) and Municipal (119). All of them received the ASC scale Frydenberg and Lewis (1997). There were significant differences between the types of Educational Establishments, with the individual establishment funded which showed greater variety of coping strategies used. Implications are discussed in light of the results found.

Trypanosoma cruzi lineages detected in congenitally infected infants and Triatoma infestans from the same disease-endemic region under entomologic surveillance in Paraguay.

Trypanosoma cruzi II is associated with Chagas disease in the southern part of South America. We analyzed T. cruzi variants in field-collected triatomines and congenitally infected infants living in the same disease-endemic region in Paraguay. Results of polymerase chain reactions for T. cruzi kinetoplast DNA and satellite DNA were positive in 83 triatomine feces samples and 58 infant blood samples. However, lineages were detected in 33 and 38 samples, respectively. Trypanosoma cruzi genotypes were determined in 56 (97%) blood samples after hybridization by using specific probes. The Tc I genotype was not detected. The prevalent sublineage was Tc IId in triatomines (27 of 33) and infant blood (36 of 58) as assessed by amplification of the 24Salpha ribosomal RNA and the mini-exon region genes. The Tc IIc genotype was detected in 20 infant blood samples and in 1 triatomine. This study shows T. cruzi II is the predominant lineage circulating in triatomines and humans in endemic areas of eastern region of Paraguay.

Early diagnosis of congenital Trypanosoma cruzi infection, using shed acute phase antigen, in Ushuaia, Tierra del Fuego, Argentina.

Chagas' disease, or American trypanosomiasis, is caused by the protozoan parasite Trypanasoma cruzi. It is estimated that 15,000 new cases of congenital T. cruzi transmission occur in the Americas each year. The aim of this study was to estimate the rate of congenital T. cruzi infection in infants born to infected women living in Ushuaia, Argentina, as well to assess a serologic test using Shed Acute Phase Antigen (SAPA) for a timely diagnosis of congenital infection. The rate of congenital infection among children in the study was 4.4% (3/68). Our results show that for infants younger than 30 days of age, matched blood samples from mother and infant were capable of identifying congenital transmission of infection using an enzyme-linked immunosorbent assay with SAPA. For infants older than 3 months, congenital infection could be ruled out using the same procedure.

[Implementation and evaluation of a locally sustainable system of prenatal diagnosis to detect cases of congenital Chagas disease in endemic areas of Paraguay]. / Implementación y evaluación de un sistema localmente sustentable de diagnostico prenatal que permite detectar casos de transmisión congénita de la enfermedad de Chagas en zonas endémicas del Paraguay.

A locally sustainable system of prenatal screening of Trypanosoma cruzi infection has been implemented in rural health care centers of endemic areas in Paraguay A total of 61.091 women from Paraguari and Cordillera Departments were serologically evaluated, where 7.802 (12,7%) resulted to be anti-T. cruzi IgG positive. A total of 1,865 infants born to seropositive mothers were examined by parasitological techniques, such as direct microscopic observation and polymerase chain reaction, and serologically by ELISA, ELISA-SAPA and IFI. 104 infected babies were detected and treated with benznidazole. The recovery of babies born to seropositive mothers performing a single examination at the age of 6 months was significantly higher, as compared with the recommended method involving two examinations, both at birth and after 6 months of age. Although at 6 months of age in 7% of the infants maternal IgG was still detected. PCR was the most sensitive technique for early detection of T. cruzi infection in babies, but we do not recommend it use for diagnosis in high endemic areas, considering that for the screening of 815 babies, 2000 reactions were needed. We propose a strategy to detect congenital transmission of Chagas disease, based on a large-scale study, where the shortcomings of the different serological and parasitological techniques are discussed.