Sensitive and specific detection of Trypanosoma cruzi DNA in clinical specimens using a multi-target real-time PCR approach

Background: The laboratory diagnosis of Chagas disease is challenging because the usefulness of different diagnostic tests will depend on the stage of the disease. Serology is the preferred method for patients in the chronic phase, whereas PCR can be successfully used to diagnose acute and congenital cases. Here we present data using a combination of three TaqMan PCR assays to detect T. cruzi DNA in clinical specimens. Methods/Principal Findings: Included in the analysis were DNA extracted from 320 EDTA blood specimens, 18 heart tissue specimens, 6 umbilical cord blood specimens, 2 skin tissue specimens and 3 CSF specimens. For the blood specimens both whole blood and buffy coat fraction were analyzed. The specimens were from patients living in the USA, with suspected exposure to T. cruzi through organ transplantation, contact with triatomine bugs or laboratory accidents, and from immunosuppressed patients with suspected Chagas disease reactivation. Real-time PCR was successfully used to diagnose acute and Chagas disease reactivation in 20 patients, including one case of organ-transmitted infection and one congenital case. Analysis of buffy coat fractions of EDTA blood led to faster diagnosis in six of these patients compared to whole blood analysis. The three real-time PCR assays produced identical results for 94% of the specimens. The major reason for discrepant results was variable sensitivity among the assays, but two of the real-time PCR assays also produced four false positive results. Conclusions/Significance: These data strongly indicate that at least two PCR assays with different performances should be combined to increase the accuracy. This evaluation also highlights the benefit of extracting DNA from the blood specimen's buffy coat to increase the sensitivity of PCR analysis.Fil: Qvarnstrom, Yvonne. Centers for Disease Control and Prevention; Estados UnidosFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Veron, Vincent. Centre hospitalier Andrée-Rosemon; Guayana FrancesaFil: Aznar, Christine. Centre hospitalier Andrée-Rosemon; Guayana FrancesaFil: Steurer, Francis. Centers for Disease Control and Prevention; Estados UnidosFil: da Silva, Alexandre J.. Centers for Disease Control and Prevention; Estados Unido

CAMBIOS EN LA PÉRDIDA DE PESO Y LA COMPOSICIÓN CORPORAL CON DIETA CETOGÉNICA Y PRACTICA DE ACTIVIDAD FÍSICA: REVISIÓN NARRATIVA, METODOLÓGICA Y SISTEMÁTICA

Introducción: la práctica de actividad física y el seguimiento de una dieta cetogénica pueden suponer un doble efecto con mejores resultados en los procesos de pérdida de peso y mejora de la composición corporal y perfil lipídico. Objetivo: el objetivo de esta revisión fue investigar los trabajos realizados con pacientes obesos que siguen una dieta cetogénica y un programa de ejercicio físico, así como calcular el tamaño del efecto en cuanto a las mejoras en la masa grasa, a través de un metaanálisis. Métodos: la selección de estudios se basó en los siguientes criterios: estudios experimentales; a) estudios experimentales (diseños controlados aleatorizados) y cuasi-experimentales (por ejemplo: pre-test/post-test); b) estudios con dieta baja en carbohidratos (< 30%) o muy baja en carbohidratos (5-10%) ( 35%); c) se admitieron estudios exclusivamente con sujetos que padecieran sobrepeso u obesidad (IMC > 25 y/o enfermedad metabólica relacionada; y d) con mediciones de composición corporal y/o perfil lipídico al principio y al final de la intervención. Resultados: se analizaron 7 artículos y 3 revisiones. Comparando los diferentes tipos de ejercicio se podría afirmar que destaca la disminución de masa muscular en aquellos en los que las intervenciones son con ejercicio aeróbico, manteniéndose e incluso aumentando, en los estudios donde se realizó un ejercicio de fuerza. El metaanálisis nos muestra una reducción significativa de la masa grasa con una heterogeneidad media, por lo tanto, habrá mayor reducción de masa grasa en grupos que realizan dieta baja en carbohidratos y ejercicio que en los que no realizan dieta o tan solo realizan ejercicio. Conclusiones: la combinación de dieta cetogénica y ejercicio físico puede reducir la masa grasa en comparación con realizar solo dieta o solo ejercicio físico. Key words: Ketogenic diet. Physical activity. Endurance. Resistance. Weight loss. Fat mass. Abstract Introduction: practice of physical activity and the ketogenic diet monitoring can have a doubAbstract Introduction: practice of physical activity and the ketogenic diet monitoring can have a double effect in helping in processes of weight loss and improvement of body composition and lipid profile. Objective: the objective of this review was to investigate the work done with obese patients who undertook a ketogenic diet and a physical exercise program, as well as to calculate the overall effect size in terms of improvements in fat mass, through a meta-analysis. Methods: the selection of studies was based on the following criteria: experimental studies; a) experimental studies (randomized controlled designs) and quasi-experimental (e.g. pre-test/post-test); b) studies with low-carbohydrate diet (< 30%) or very low in carbohydrates (5-10%) ( 35%); c) studies were admitted exclusively with subjects that facility overweight or obesity (BMI > 25; and d) with measurements of body composition and/or Lipid profile at the beginning and end of the intervention. Results: for the methodological review, 7 articles and 3 reviews were analyzed. All studies, whether by establishing aerobic or strength training and show significant weight loss in all outcomes. Conclusions: comparing different types of exercise, we could say that interventions based on endurance exercise reported a decrease in muscle mass, however there was a maintenance, and even an increase, in studies with resistance exercises. Meta-analysis showed significant results at the global level with a medium heterogeneity, therefore, there will be greater reduction of fat mass in groups that realize diets with low carbohydrates and exercise that in those who do not undertake this type of diet, and those only perform exercise

International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients

A century after its discovery, Chagas disease, caused by the parasite Trypanosoma cruzi, still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The polymerase chain reaction (PCR) has been proposed as a sensitive laboratory tool for detection of T. cruzi infection and monitoring of parasitological treatment outcome. However, high variation in accuracy and lack of international quality controls has precluded reliable applications in the clinical practice and comparisons of data among cohorts and geographical regions. In an effort towards harmonization of PCR strategies, 26 expert laboratories from 16 countries evaluated their current PCR procedures against sets of control samples, composed by serial dilutions of T.cruzi DNA from culture stocks belonging to different lineages, human blood spiked with parasite cells and blood samples from Chagas disease patients. A high variability in sensitivities and specificities was found among the 48 reported PCR tests. Out of them, four tests with best performance were selected and further evaluated. This study represents a crucial first step towards device of a standardized operative procedure for T. cruzi PCR.Fil: Schijman, Alejandro G. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET). Laboratorio de Biología Molecular de la Enfermedad de Chagas (LabMECh); Argentina.Fil: Bisio, Margarita. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET). Laboratorio de Biología Molecular de la Enfermedad de Chagas (LabMECh); Argentina.Fil: Orellana, Liliana. Universidad de Buenos Aires. Instituto de Cálculo; Argentina.Fil: Sued, Mariela. Universidad de Buenos Aires. Instituto de Cálculo; Argentina.Fil: Duffy, Tomás. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET). Laboratorio de Biología Molecular de la Enfermedad de Chagas (LabMECh); Argentina.Fil: Mejia Jaramillo, Ana M. Universidad de Antioquia. Grupo Chagas; Colombia.Fil: Cura, Carolina. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET). Laboratorio de Biología Molecular de la Enfermedad de Chagas (LabMECh); Argentina.Fil: Auter, Frederic. French Blood Services; Francia.Fil: Veron, Vincent. Universidad de Parasitología. Laboratorio Hospitalario; Guayana Francesa.Fil: Qvarnstrom, Yvonne. Centers for Disease Control. Department of Parasitic Diseases; Estados Unidos.Fil: Deborggraeve, Stijn. Institute of Tropical Medicine; Bélgica.Fil: Hijar, Gisely. Instituto Nacional de Salud; Perú.Fil: Zulantay, Inés. Facultad de Medicina; Chile.Fil: Lucero, Raúl Horacio. Universidad Nacional del Nordeste; Argentina.Fil: Velázquez, Elsa. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología Dr. Mario Fatala Chaben; Argentina.Fil: Tellez, Tatiana. Universidad Mayor de San Simon. Centro Universitario de Medicina Tropical; Bolivia.Fil: Sanchez Leon, Zunilda. Universidad Nacional de Asunción. Instituto de Investigaciones en Ciencias de la Salud; Paraguay.Fil: Galvão, Lucia. Faculdade de Farmácia; Brasil.Fil: Nolder, Debbie. Hospital for Tropical Diseases. London School of Tropical Medicine and Hygiene Department of Clinical Parasitology; Reino Unido.Fil: Monje Rumi, María. Universidad Nacional de Salta. Laboratorio de Patología Experimental; Argentina.Fil: Levi, José E. Hospital Sirio Libanês. Blood Bank; Brasil.Fil: Ramirez, Juan D. Universidad de los Andes. Centro de Investigaciones en Microbiología y Parasitología Tropical; Colombia.Fil: Zorrilla, Pilar. Instituto Pasteur; Uruguay.Fil: Flores, María. Instituto de Salud Carlos III. Centro de Mahahonda; España.Fil: Jercic, Maria I. Instituto Nacional De Salud. Sección Parasitología; Chile.Fil: Crisante, Gladys. Universidad de los Andes. Centro de Investigaciones Parasitológicas J.F. Torrealba; Venezuela.Fil: Añez, Néstor. Universidad de los Andes. Centro de Investigaciones Parasitológicas J.F. Torrealba; Venezuela.Fil: De Castro, Ana M. Universidade Federal de Goiás. Instituto de Patologia Tropical e Saúde Pública (IPTSP); Brasil.Fil: Gonzalez, Clara I. Universidad Industrial de Santander. Grupo de Inmunología y Epidemiología Molecular (GIEM); Colombia.Fil: Acosta Viana, Karla. Universidad Autónoma de Yucatán. Departamento de Biomedicina de Enfermedades Infecciosas y Parasitarias Laboratorio de Biología Celular; México.Fil: Yachelini, Pedro. Universidad Católica de Santiago del Estero. Instituto de Biomedicina; Argentina.Fil: Torrico, Faustino. Universidad Mayor de San Simon. Centro Universitario de Medicina Tropical; Bolivia.Fil: Robello, Carlos. Instituto Pasteur; Uruguay.Fil: Diosque, Patricio. Universidad Nacional de Salta. Laboratorio de Patología Experimental; Argentina.Fil: Triana Chavez, Omar. Universidad de Antioquia. Grupo Chagas; Colombia.Fil: Aznar, Christine. Universidad de Parasitología. Laboratorio Hospitalario; Guayana Francesa.Fil: Russomando, Graciela. Universidad Nacional de Asunción. Instituto de Investigaciones en Ciencias de la Salud; Paraguay.Fil: Büscher, Philippe. Institute of Tropical Medicine; Bélgica.Fil: Assal, Azzedine. French Blood Services; Francia.Fil: Guhl, Felipe. Universidad de los Andes. Centro de Investigaciones en Microbiología y Parasitología Tropical; Colombia.Fil: Sosa Estani, Sergio. ANLIS Dr.C.G.Malbrán. Centro Nacional de Diagnóstico e Investigación en Endemo-Epidemias; Argentina.Fil: DaSilva, Alexandre. Centers for Disease Control. Department of Parasitic Diseases; Estados Unidos.Fil: Britto, Constança. Instituto Oswaldo Cruz/FIOCRUZ. Laboratório de Biologia Molecular e Doenças Endêmicas; Brasil.Fil: Luquetti, Alejandro. Laboratório de Pesquisa de Doença de Chagas; Brasil.Fil: Ladzins, Janis. World Health Organization (WHO). Special Programme for Research and Training in Tropical Diseases (TDR); Suiza

International Study to Evaluate PCR Methods for Detection of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients

A century after its discovery, Chagas disease, caused by the parasite Trypanosoma cruzi, still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The polymerase chain reaction (PCR) has been proposed as a sensitive laboratory tool for detection of T. cruzi infection and monitoring of parasitological treatment outcome. However, high variation in accuracy and lack of international quality controls has precluded reliable applications in the clinical practice and comparisons of data among cohorts and geographical regions. In an effort towards harmonization of PCR strategies, 26 expert laboratories from 16 countries evaluated their current PCR procedures against sets of control samples, composed by serial dilutions of T.cruzi DNA from culture stocks belonging to different lineages, human blood spiked with parasite cells and blood samples from Chagas disease patients. A high variability in sensitivities and specificities was found among the 48 reported PCR tests. Out of them, four tests with best performance were selected and further evaluated. This study represents a crucial first step towards device of a standardized operative procedure for T. cruzi PCR

The triatominae species of French Guiana (Heteroptera: Reduviidae)

An annotated list of the triatomine species present in French Guiana is given. It is based on field collections carried out between 1993-2008, museum collections and a literature review. Fourteen species, representing four tribes and six genera, are now known in this country and are illustrated (habitus). Three species are recorded from French Guiana for the first time: Cavernicola pilosa, Microtriatoma trinidadensis and Rhodnius paraensis. The two most common and widely distributed species are Panstrongylus geniculatus and Rhodnius pictipes. The presence of two species (Panstrongylus megistus and Triatoma maculata) could be fortuitous and requires confirmation. Also, the presence of Rhodnius prolixus is doubtful; while it was previously recorded in French Guiana, it was probably mistaken for R. robustus. A key for French Guiana's triatomine species is provided

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

Background: Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR).Methods/Principal Findings: The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm.Conclusions/Significance: Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.This work received financial support from the Ministry of Science and Technology of Argentina [PICT 2011-0207 to AGS] and the National Scientific and Technical Research Council in Argentina (CONICET) [PIP 112 2011-010-0974 to AGS]. Work related to evaluation of biological samples was partially sponsored by the Pan-American Health Organization (PAHO) [Small Grants Program PAHO-TDR]; the Drugs and Neglected Diseases Initiative (DNDi, Geneva, Switzerland), Wellcome Trust (London, United Kingdom), SANOFI-AVENTIS (Buenos Aires, Argentina) and the National Council for Science and Technology in Mexico (CONACYT) [FONSEC 161405 to JMR]

La administración electrónica como herramienta de inclusión digital

¿Puede ser la administración electrónica una herramienta de inclusión digital? ¿Qué puede aportar la administración electrónica para avanzar en la inclusión digital? ¿Qué podemos hacer, cada uno desde nuestra actividad, para favorecer la inclusión digital? Estas son algunas de las preguntas que se formulaban los participantes en las III Jornadas sobre Derecho y Tecnología así como al XI Encuentro de Gobierno Electrónico e Inclusión Digital, celebrados en Zaragoza los días 23 y 24 de mayo de 2011. Cada uno, desde su perspectiva, trató de aportar ideas en esa línea que marcamos en la convocatoria del evento: «La administración electrónica como herramienta de inclusión digital». Estas aportaciones están recogidas en este libro, agrupadas en tres bloques diferentes. En el primero, bajo el título Políticas de inclusión digital desde la perspectiva de la administración electrónica, tienen cabida aquellas reflexiones sobre qué hacer para favorecer la inclusión digital desde la administración electrónica, con enfoques concretos en Brasil o Iberoamérica, o de tipo general, aplicable en cualquier país. En el segundo, el título Casos reales de inclusión digital desde la perspectiva de la administración electrónica reúne experiencias concretas llevadas a cabo en España y Brasil. Por último, y no menos importante, el título Inclusión digital desde las aulas universitarias recoge propuestas para fomentar la inclusión digital desde las aulas universitarias

Global matrix 4.0 physical activity report card grades for children and adolescents : results and analyses from 57 countries

Background: The Global Matrix 4.0 on physical activity (PA) for children and adolescents was developed to achieve a comprehensive understanding of the global variation in children’s and adolescents’ (5–17 y) PA, related measures, and key sources of influence. The objectives of this article were (1) to summarize the findings from the Global Matrix 4.0 Report Cards, (2) to compare indicators across countries, and (3) to explore trends related to the Human Development Index and geo-cultural regions. Methods: A total of 57 Report Card teams followed a harmonized process to grade the 10 common PA indicators. An online survey was conducted to collect Report Card Leaders’ top 3 priorities for each PA indicator and their opinions on how the COVID-19 pandemic impacted child and adolescent PA indicators in their country. Results: Overall Physical Activity was the indicator with the lowest global average grade (D), while School and Community and Environment were the indicators with the highest global average grade (C+). An overview of the global situation in terms of surveillance and prevalence is provided for all 10 common PA indicators, followed by priorities and examples to support the development of strategies and policies internationally. Conclusions: The Global Matrix 4.0 represents the largest compilation of children’s and adolescents’ PA indicators to date. While variation in data sources informing the grades across countries was observed, this initiative highlighted low PA levels in children and adolescents globally. Measures to contain the COVID-19 pandemic, local/international conflicts, climate change, and economic change threaten to worsen this situation

Histoplasma capsulatum et environnement en Guyane française (études préliminaires)

L'agent de l'histoplasmose, Histoplasma capsulatum, est un champignon dimorphique, endémique dans de nombreuses régions tropicales. En Guyane française, les premiers isolements environnementaux de ce pathogène ont été réalisés en 1955 du sol d un poulailler. Depuis, aucune autre étude écologique n a été réalisée du fait de l important manque de sensibilité des techniques historiques d isolement. L objectif de ce travail était d effectuer une première étude de l intérêt de coupler la méthode de PCR en temps réel développée à l hôpital de Cayenne à un kit dédié de purification d ADN pour la recherche d H. capsulatum dans l environnement. Les échantillons analysés ont été collectés en 2002 et 2008 dans des sites propices à la contamination humaine et animale et localisés en zone urbaine et forestière. Dans une phase préliminaire, seul le kit PowerSoil® a permis la mise en évidence de l ADN cible dans des témoins positifs et a donc été retenu. L étude rétrospective de 30 échantillons collectés en 2002 et conservés à température ambiante n a pas été concluante. L étude prospective réalisée en 2008 a montré que 11.76 % des prélèvements urbains collectés étaient positifs pour l ADN d H. capsulatum. L examen microscopique attentif de ces prélèvements a permis la visualisation d éléments évoquant les chlamydospores d H. capsulatum. Un seul isolement du champignon en culture a cependant été possible. Les autres milieux ont été rapidement envahis par des contaminants. Ces premiers résultats démontrent l intérêt de la PCR en temps réel pour la recherche environnementale de champignons pathogènes. Ils prouvent de plus la présence d H. capsulatum en milieu urbain en Guyane françaiseThe causative agent of histoplasmosis, Histoplasma capsulatum, is a dimorphic fungus that is endemic in many tropical areas. In French Guiana, H. capsulatum was isolated in 1955 from soil samples collected in a chicken farm. Since then no other ecologic studies were performed, mostly due to the important lack of sensitivity of classic culture methods. The purpose of this study was to use the real-time PCR method developed in Cayenne hospital with a DNA purification kit to detect H. capsulatum in the environment. The retrospective analysis of 30 samples collected in 2002 and kept at ambient temperature was inconclusive. The prospective analysis of samples collected in 2008 in urban areas showed that 11.76% of them were positive for H. capsulatum DNA. The meticulous direct microscopic examination of these PCR positive samples showed constantly the presence of tuberculated structures evocating H. capsulatum. However, attempts to isolate the fungus by culture were only successful in one case. All other plates were rapidly overgrown by contaminants. These first results proved the presence of H. capsulatum in urban environment in French Guiana, and the interesting role of molecular methods to screen soil samples for pathogenic fungi.AMIENS-BU Santé (800212102) / SudocSudocFranceF