Development of vaccines for Chagas disease (CRUZIVAX): stakeholders' preferences and potential impacts on healthcare.

A vaccine for Chagas disease does not currently exist. This study aims to inform the development of two vaccines for the prevention and treatment of Trypanosoma cruzi infection, and guide their pre-clinical phase up to clinical phase I. The three main objectives are: 1) to explore patients' and policy makers' preferences on the candidate vaccines in Argentina and Spain; 2) to investigate health-related quality of life of patients affected by Chagas disease; and 3) to assess the potential health provider savings associated with the vaccines, in terms of resource use and health care costs. Discrete choice experiments will be employed to estimate and characterize the theoretical demand for the vaccines and investigate patients' and policy makers' preferences. Health-related quality of life will be assessed using the EQ-5D-3L questionnaire. Resources use and costs associated with Chagas disease will be investigated using information from the databases of the Hospital Clínic of Barcelona.

Development of vaccines for Chagas disease (CRUZIVAX): stakeholders’ preferences and potential impacts on healthcare / Desarrollo de vacunas para la enfermedad de Chagas (CRUZIVAX): preferencias de las partes interesadas y posibles impactos en la asistencia sanitaria

A vaccine for Chagas disease does not currently exist. This study aims to inform the development of two vaccines for the prevention and treatment of Trypanosoma cruzi infection, and guide their pre-clinical phase up to clinical phase I. The three main objectives are: 1) to explore patients’ and policy makers’ preferences on the candidate vaccines in Argentina and Spain; 2) to investigate health-related quality of life of patients affected by Chagas disease; and 3) to assess the potential health provider savings associated with the vaccines, in terms of resource use and health care costs. Discrete choice experiments will be employed to estimate and characterize the theoretical demand for the vaccines and investigate patients’ and policy makers’ preferences. Health-related quality of life will be assessed using the EQ-5D-3L questionnaire. Resources use and costs associated with Chagas disease will be investigated using information from the databases of the Hospital Clínic of Barcelona. (AU)

No existen vacunas para la enfermedad de Chagas. Este trabajo pretende informar la fase preclínica de dos vacunas para la prevención y el tratamiento de la infección por Trypanosoma cruzi. Los objetivos principales son tres: 1) investigar las preferencias de los pacientes y de los responsables de políticas sanitarias en Argentina y España; 2) investigar la calidad de vida relacionada con la salud de los pacientes afectados por la enfermedad de Chagas; y 3) estimar los ahorros potenciales asociados con las vacunas para los proveedores de salud. Se usarán experimentos de elección discreta para estimar y caracterizar la demanda teórica de las vacunas e investigar las preferencias de los pacientes y de los responsables de las políticas sanitarias. La calidad de vida relacionada con la salud se evaluará mediante el cuestionario EQ-5D-3L. Se investigarán el uso de recursos y los costes asociados a la enfermedad de Chagas utilizando bases de datos del Hospital Clínic de Barcelona. (AU)

Urban infestation by Triatoma infestans (Hemiptera: Reduviidae), an overlooked phenomena for Chagas disease in Argentina.

Vector-borne transmission of Chagas disease in urban areas of Argentina has been an overlooked phenomena. We conducted the first comprehensive cross-sectional study of domestic infestation with Triatoma infestans and vector infection with Trypanosoma cruzi in a metropolitan area of San Juan, Argentina. Our results document the occurrence of T. infestans infected with T. cruzi in human sleeping quarters. In this urban setting, we also show that infestation was associated with construction materials, the presence of chickens, cats and a large number of dogs that can provide blood meals for the vector. Our findings reveal new challenges for vectorial control agencies.

Update process ofthe National Agenda for Public Health Research 2021-2022 / Proceso de actualización de la agenda nacional de investigación en salud pública 2021-2022

RESUMEN INTRODUCCIÓN : Una Agenda Nacional de Investigación en Salud Pública (ANISP) participativa y con priorización temática constituye un elemento estratégico para generar recomendaciones y políticas públicas basadas en evidencia, que imparten positivamente en la salud de las poblaciones y permitan lograr los objetivos sanitarios. En la actualización de la ANISP participaron la Dirección de Investigación en Salud (DIS) del Ministerio de Salud de la Nación (MSAL), a través de la Red Ministerial de Investigación en Salud (REMINSA), y actores de los niveles gubernamentales provinciales y nacionales pertenecientes a los sectores público, privado, de la salud, académico y de investigación. Se adaptó la herramienta original propuesta por la Organización Panamericana de la Salud, utilizada en el proceso en 2019. La actualización abarcó diferentes etapas. La selección de los temas contó con la legitimidad, reconocimiento y participación de los actores vinculados a la salud, a la gestión gubernamental y privada y a la investigación científica; se trabajó de manera federal y transversal, por consenso con las redes provinciales y un Comité Central Asesor en el MSAL. A partir de los lineamientos preliminares obtenidos, se elaboró una encuesta en línea semiestructurada, que fue distribuida a todos los actores federales y recibió 431 respuestas. El proceso resultó en 55 lineamientos priorizados, divididos en 6 áreas temáticas y 33 subtemas, seleccionados por votación según importancia, impacto y factibilidad.

ABSTRACT INTRODUCTION : A participatory National Public Health Research Agenda (ANISP) with thematic prioritization is a strategic element to generóte evidence-based recommendations and public policies that have a positive impact on the health of populations and enable to achieve health objectives. The Directorate of Health Research (DIS) ofthe Argentine Ministry of Health (MSAL), through the Ministerial NetWork of Health Research (REMINSA), along with adors from the provincial and national government levels belonging to public, privóte, health, academic and research sectors participated in the update of the ANISP. They adapted the original tooI proposed by the Pan American Health Organizatlon and used in the process in 2019. The update included different stages. The selection ofthe topics had the legitimacy, recognition and participation ofthe actors involved, related to health, to government and privóte management and to scientific research; the work was conducted in a federal and transversal manner by consensus with the provincial networks and a Central Advisory Committee in the MSAL. Based on the preliminary guidelines obtained, a semi-structured online survey was developed and distributed to all federal actors, receiving 431 responses. The process resulted in 55 priorilized guidelines, divided into 6 thematic oreas and 33 sub-themes, selected by voting according to importance, impact and feasibility.

Proceso de actualización de la Agenda Nacional de Investigación Salud Pública 2021-2022 / Update process of the National Agenda for Public Health Research 2021-2022

INTRODUCCIÓN: Una Agenda Nacional de Investigación en Salud Pública (ANISP) participativa y con priorización temática constituye un elemento estratégico para generar recomendaciones y políticas públicas basadas en evidencia, que impacten positivamente en la salud de las poblaciones y permitan lograr los objetivos sanitarios. En la actualización de la ANISP participaron la Dirección de Investigación en Salud (DIS) del Ministerio de Salud de la Nación (MSAL), a través de la Red Ministerial de Investigación en Salud (REMINSA), y actores de los niveles gubernamentales provinciales y nacionales pertenecientes a los sectores público, privado, de la salud, académico y de investigación. Se adaptó la herramienta original propuesta por la Organización Panamericana de la Salud, utilizada en el proceso en 2019. La actualización abarcó diferentes etapas. La selección de los temas contó con la legitimidad, reconocimiento y participación de los actores vinculados a la salud, a la gestión gubernamental y privada y a la investigación científica; se trabajó de manera federal y transversal, por consenso con las redes provinciales y un Comité Central Asesor en el MSAL. A partir de los lineamientos preliminares obtenidos, se elaboró una encuesta en línea semiestructurada, que fue distribuida a todos los actores federales y recibió 431 respuestas. El proceso resultó en 55 lineamientos priorizados, divididos en 6 áreas temáticas y 33 subtemas, seleccionados por votación según importancia, impacto y factibilidad

Urban infestation by Triatoma infestans (Hemiptera: Reduviidae), an overlooked phenomena for Chagas disease in Argentina

Vector-borne transmission of Chagas disease in urban areas of Argentina has been an overlooked phenomena. We conducted the first comprehensive cross-sectional study of domestic infestation with Triatoma infestans and vector infection with Trypanosoma cruzi in a metropolitan area of San Juan, Argentina. Our results document the occurrence of T. infestans infected with T. cruzi in human sleeping quarters. In this urban setting, we also show that infestation was associated with construction materials, the presence of chickens, cats and a large number of dogs that can provide blood meals for the vector. Our findings reveal new challenges for vectorial control agencies.

Resistance to deltamethrin in Triatoma infestans: microgeographical distribution, validation of a rapid detection bioassay and evaluation of a fumigant canister as control alternative strategy.

Triatoma infestans (Klug) (Hemiptera: Reduviidae) is the main vector of Chagas disease in the Southern Cone of America and resistance to pyrethroid insecticides has been detected in several areas from its geographical distribution. Pyrethroid resistance presents a complex geographical pattern at different spatial scales. However, it is still unknown if the toxicological variability is a common feature within villages of the Gran Chaco were high resistance was descripted. The objectives of this study were to determine: (a) the microgeographical distribution of the deltamethrin-resistance in insects from Pampa Argentina village, (b) the performance of the insecticide impregnated paper bioassay to evaluate deltamethrin-resistance in field collected insects and (c) the lethal activity of the fumigant canister containing DDVP against insects resistant to deltamethrin. High survival of T. infestans exposed to discriminant dose was observed in the samples of all the evaluated dwellings, suggesting that the resistance to deltamethrin is homogeneous at the microgeographical level. Resistance determination by impregnated paper bioassay was similar to traditional topical determination, highlighting the use of this rapid methodology in field large-scale monitoring. The fumigant canister was not effective against resistant insects, remarking the need to develop suitable formulations that ensure minimal toxicological risk and high effectivity.

Human infectiousness and parasite load in chronic patients seropositive for Trypanosoma cruzi in a rural area of the Argentine Chaco.

A key parameter in the transmission of vector-borne infections, including Chagas disease, is the ability of the different host species to transmit the parasite to the vector (infectiousness). Here, we determined infectiousness to the vector of Trypanosoma cruzi-seropositive humans examined by artificial xenodiagnosis (XD), established its relationship with T. cruzi DNA levels (a surrogate of intensity of parasitemia) quantified by real-time PCR (qPCR), and assessed whether infectiousness was associated with the body mass index (BMI), age, ethnic background and parasite genotype. XD was performed to 117 T. cruzi-seropositive residents from Pampa del Indio and parasite load was quantified in 81 of them. Using optical microscopy (OM) 33.6% of the seropositive people tested were infectious and this fraction nearly doubled (66.0%) when XD triatomines were examined by kDNA-PCR. The mean infectiousness (defined as the percentage of all infected triatomines detected by OM at any time point among the total number of insects examined by OM 30 days post-feeding) was 5.2%, and the mean parasite load was 0.51 parasite equivalents per ml. Infectiousness to the vector was associated negatively with age and BMI, and positively with the detection of parasitemia by kDNA-PCR, and parasite load by qPCR in bivariate analysis. Patients with a positive XD by OM exhibited a significantly higher mean parasite load. Using multiple regression, infectiousness was associated with parasite load (positively) and with the household presence of T. infestans and Qom ethnic group (negatively); no significant association was observed with age or its interaction with ethnicity. We did not find significant associations between identified DTUs and infectiousness or parasite load. Infectiousness was aggregated: 18% of the people examined by XD generated 80% of the infected triatomines. Detecting and treating the super-infectious fraction of the infected human would disproportionally impact on domestic transmission risks. Nonetheless, treatment of all eligible infected people who meet the inclusion criteria regardless of their parasitemia should be ensured to improve their prognosis.

Human Trypanosoma cruzi infection is driven by eco-social interactions in rural communities of the Argentine Chaco.

The transmission of Trypanosoma cruzi to humans is determined by multiple ecological, socio-economic and cultural factors acting at different scales. Their effects on human infection with T. cruzi have often been examined separately or using a limited set of ecological and socio-demographic variables. Herein, we integrated the ecological and social dimensions of human infection risk with the spatial distribution patterns of human and vector (Triatoma infestans) infection in rural communities of the Argentine Chaco composed of indigenous people (90% Qom) and a creole minority. We conducted serosurveys in 470 households aiming at complete population enumeration over 2012-2015. The estimated seroprevalence of T. cruzi prior to the implementation of an insecticide spraying campaign (2008) was 29.0% (N = 1,373 in 301 households), and was twice as large in Qom than creoles. Using generalized linear mixed models, human seropositive cases significantly increased with infected triatomine abundance, having a seropositive household co-inhabitant and household social vulnerability (a multidimensional index of poverty), and significantly decreased with increasing host availability in sleeping quarters (an index summarizing the number of domestic hosts for T. infestans). Vulnerable household residents were exposed to a higher risk of infection even at low infected-vector abundances. The risk of being seropositive increased significantly with house infestation among children from stable households, whereas both variables were not significantly associated among children from households exhibiting high mobility within the communities, possibly owing to less consistent exposures. Human infection was clustered by household and at a larger spatial scale, with hotspots of human and vector infection matching areas of higher social vulnerability. These results were integrated in a risk map that shows high-priority areas for targeted interventions oriented to suppress house (re)infestations, detect and treat infected children, and thus reduce the burden of future disease.

High levels of human infection with Trypanosoma cruzi associated with the domestic density of infected vectors and hosts in a rural area of northeastern Argentina.

BACKGROUND: Insecticide spraying campaigns designed to suppress the principal vectors of the Chagas disease usually lack an active surveillance system that copes with house reinvasion. Following an insecticide campaign with no subsequent surveillance over a 12-year period, we implemented a longitudinal intervention programme including periodic surveys for Triatoma infestans, full-coverage house spraying with insecticides, and selective control in a well-defined rural area of the Argentinean Chaco inhabited by Creoles and one indigenous group (Qom). Here, we conducted a cross-sectional study and report the age-specific seroprevalence of human T. cruzi infection by group, and examine the association between human infection, the onset of the intervention, the relative density of infected domestic bugs, and the household number of infected people, dogs, or cats. RESULTS: The seroprevalence of infection among 691 residents examined was 39.8% and increased steadily with age, reaching 53-70% in those older than 20 years. The mean annual force of infection was 2.5 per 100 person-years (95% CI: 1.8-3.3%). Infection in children younger than 16 years born before the intervention programme was two to four times higher in houses with infected T. infestans than in houses without them and was six times higher when there were both infected dogs or cats and bugs than when they were absent. The model-averaged estimate of the intervention effect suggests that the odds of seropositivity were about nine times smaller for those born after the onset of the intervention than for those born before it, regardless of ethnic background, age, gender, household wealth, and cohabitation with T. cruzi-infected vectors or human hosts. Human infection was also closely associated with the baseline abundance of infected domestic triatomines and the number of infected cohabitants. Two of 43 children born after interventions were T. cruzi-seropositive; since their mothers were seropositive and both resided in apparently uninfested houses they were attributed to vertical transmission. Alternatively, these cases could be due to non-local vector-borne transmission. CONCLUSIONS: Our study reveals high levels of human infection with T. cruzi in the Argentinean Chaco, and the immediate impact of sustained vector surveillance and selective control actions on transmission.

Factores de riesgo en Chagas urbano / Risk factors in urban Chagas

San Juan presenta una alta infestación por Triatoma infestans en áreas urbanas, por ello nos propusimos estudiar los factores de riesgo asociados con la transmisión vectorial del T. cruzi en ámbito urbano del Departamento Rawson. Se empleo un diseño observacional y transversal de enfoque mixto; cuali-cuantitativo. Se encuestó sobre las características de la vivienda, de movilidad, de vulnerabilidad socio-sanitarias y de conocimientos de la enfermedad. Se realizaron encuestas entomológicas en las viviendas y serológicas en la población y en los perros. Mediante modelos multinivel se evaluó la importancia relativa de los factores para explicar la seropositividad a T. cruzi y la infestación por Triatoma infestans. Se realizó análisis espacial para estudiar la distribución de la infección e infestación. Se llevaron a cabo encuestas dialogadas a referentes de la comunidad y de los equipos de salud y vectores. Se evaluaron 347/758 viviendas visitadas, el 13,8% presentó infestación que se asoció al número de sitios en el peridomicilio y techos de machimbre y/o caña. Se realizó diagnóstico en 614/1057 habitantes detectándose una prevalencia del 3,09%. Se estudiaron 130 caninos de los cuales 10,0% fueron reactivos. Ninguna variable pudo explicar la infección, se registró una tendencia que asocia positivamente la vulnerabilidad con la presencia de personas infectadas. La infestación no se identificó estadísticamente agregada a nivel de manzana mientras que la infección en perro y humanos si, fenómeno observado también en el análisis espacial. De las encuestas dialogadas surge la necesidad de realizar más difusión, de promover acciones de control integrales y de generar adaptaciones a los protocolos de control vectorial. Nuestro estudio pone en evidencia la extensión y complejidad del problema de la transmisión vectorial de la enfermedad de Chagas en ámbitos urbanos e identifica grandes desafíos para la ejecución de acciones de control

Estudio microgeográfico de la susceptibilidad a Deltametrina por Triatoma Infestans en Chaco / Microgeographic study of susceptibility to Deltamethrin by Triatoma Infestans in Chaco

INTRODUCCIÓN La resistencia a insecticidas en T. infestans se evidencia como un problema complejo dado que evolucionó presentándose con distintos perfiles y mecanismos. Algunos estudios revelaron heterogeneidad toxicológica en diversas áreas de la provincia del Chaco. En ese contexto, resultó de interés investigar si existe esta heterogeneidad toxicológica entre viviendas de un mismo paraje en una zona de alta resistencia. OBJETIVO Caracterizar a nivel microgeográfico la susceptibilidad frente al insecticida piretroide Deltametrina de las vinchucas en el paraje rural Pampa Argentina. MÉTODOS Se realizó un estudio de tipo transversal descriptivo y correlacional que comprendió relevamientos entomológicos, encuestas socio-demográficas, monitoreo de la infestación de las viviendas y análisis de susceptibilidad a piretroides de insectos mediante un abordaje con participación comunitaria. RESULTADOS Se monitorearon 106 hogares; el 46.2% (n=49) presentaron infestación domiciliaria por T. infestans. Los bioensayos por tópico tomaron como base los insectos colectados de 22 viviendas hallando una mortalidad promedio del 0,85% (IC del 95%; 0,42 a 1,28). En bioensayos con papeles impregnados se expusieron 461 insectos identificándose una supervivencia de 97,6% y de mortalidad del 2.4%. Respecto a la distribución espacial de la resistencia, puede observarse que se registró una dispersión alta y distribución homogénea de insectos resistentes en toda el área de estudio. Al análisis bivariado, la Acumulación de Elementos en los dormitorios, la Presencia de Gallinas y la Manifestación de Rastros estuvieron significativamente asociados a la infestación; sin embargo no se obtuvo asociaciones de las variables respecto de la resistencia. DISCUSIÓN Existe una homogeneidad en la distribución espacial de la resistencia en el paraje monitoreado, por ello, la situación de la resistencia a piretroides se esgrime como un problema complejo que requiere la generación de nuevas estrategias de control de baja toxicidad y que sean aceptadas por la comunidad

Improving access to Chagas disease diagnosis and etiologic treatment in remote rural communities of the Argentine Chaco through strengthened primary health care and broad social participation.

BACKGROUND: Rural populations in the Gran Chaco region have large prevalence rates of Trypanosoma cruzi infection and very limited access to diagnosis and treatment. We implemented an innovative strategy to bridge these gaps in 13 rural villages of Pampa del Indio held under sustained vector surveillance and control. METHODOLOGY: The non-randomized treatment program included participatory workshops, capacity strengthening of local health personnel, serodiagnosis, qualitative and quantitative PCRs, a 60-day treatment course with benznidazole and follow-up. Parents and healthcare agents were instructed on drug administration and early detection and notification of adverse drug-related reactions (ADR). Healthcare agents monitored medication adherence and ADRs at village level. PRINCIPAL FINDINGS: The seroprevalence of T. cruzi infection was 24.1% among 395 residents up to 18 years of age examined. Serodiagnostic (70%) and treatment coverage (82%) largely exceeded local historical levels. Sixty-six (85%) of 78 eligible patients completed treatment with 97% medication adherence. ADRs occurred in 32% of patients, but most were mild and manageable. Four patients showing severe or moderate ADRs required treatment withdrawal. T. cruzi DNA was detected by qPCR in 47 (76%) patients before treatment, and persistently occurred in only one patient over 20-180 days posttreatment. CONCLUSIONS AND SIGNIFICANCE: Our results demonstrate that diagnosis and treatment of T. cruzi infection in remote, impoverished rural areas can be effectively addressed through strengthened primary healthcare attention and broad social participation with adequate external support. This strategy secured high treatment coverage and adherence; effectively managed ADRs, and provided early evidence of positive therapeutic responses.

Mesas de gestión integradas locales como estrategia para la vigilancia comunitaria de Chagas en localidades de San Juan y Chaco / Local integrated management tables as a strategy for community surveillance of Chagas in localities of San Juan and Chaco

INTRODUCCIÓN Entre las líneas estratégicas del plan Nacional de Chagas (2011-2016) se hace especial hincapié a la vigilancia comunitaria participativa y promueve la conformación y/o fortalecimiento de las mesas de gestión integrada (MGI) a nivel local para estimular la movilización social e instalar y fortalecer la vigilancia comunitaria. En este contexto el objetivo de este estudio fue "Promover y favorecer la implementación de las MGI a nivel local". MÉTODOS Se realizó un estudio descriptivo de corte transversal con un enfoque cuali y cuantitativo en dos localidades, Quitilipi (Chaco) y Ullum (San Juan). El estudio incluyó encuestas aleatorias a pobladores y entrevistas a referentes de la comunidad. Se evaluó conocimientos, actitudes, prácticas en la prevención y control de Chagas, relaciones interinstitucionales, interés por participar en la comunidad. RESULTADOS Existe un alto porcentaje de desconocimiento de la comunidad con respecto a la enfermedad, vías de transmisión y medidas de prevención. Las entrevistas realizadas revelaron un interés alto de los entrevistados en incluirse en posibles acciones en torno a la participación y la vigilancia para Chagas, sin embargo, el interés se ve debilitado cuando no se obtienen las respuestas institucionales al esfuerzo individual del referente o cuando se presentan obstáculos que generan resignación y desmotivación En ambas localidades no fue posible la creación de la mesa de gestión integrada (MGI). DISCUSIÓN Los cambios en los escenarios políticos e institucionales fueron el principal obstáculo para instalar MGI con participación activa de la comunidad. Para integrar la comunidad a estrategias de vigilancia como las MGI, primero debe haber un convencimiento y compromiso de las instituciones para incorporar a la comunidad como protagonista activo desde la planificación de las acciones y no sólo como denunciante de la presencia de vinchucas

Trans-sialidase inhibition assay detects Trypanosoma cruzi infection in different wild mammal species.

The detection of Trypanosoma cruzi infection in mammals is crucial for understanding the eco-epidemiological role of the different species involved in parasite transmission cycles. Xenodiagnosis (XD) and hemoculture (HC) are routinely used to detect T. cruzi in wild mammals. Serological methods are much more limited because they require the use of specific antibodies to immunoglobulins of each mammalian species susceptible to T. cruzi. In this study we detected T. cruzi infection by trans-sialidase (TS) inhibition assay (TIA). TIA is based on the antibody neutralization of a recombinant TS that avoids the use of anti-immunoglobulins. TS activity is not detected in the co-endemic protozoan parasites Leishmania spp and T. rangeli. In the current study, serum samples from 158 individuals of nine wild mammalian species, previously tested by XD, were evaluated by TIA. They were collected from two endemic areas in northern Argentina. The overall TIA versus XD co-reactivity was 98.7% (156/158). All 18 samples from XD-positive mammals were TIA-positive (co-positivity, 100%) and co-negativity was 98.5% (138/140). Two XD-negative samples from a marsupial (Didelphis albiventris) and an edentate (Dasypus novemcinctus) were detected by TIA. TIA could be used as a novel tool for serological detection of Trypanosoma cruzi in a wide variety of sylvatic reservoir hosts.

Diagnostic peptide discovery: prioritization of pathogen diagnostic markers using multiple features.

The availability of complete pathogen genomes has renewed interest in the development of diagnostics for infectious diseases. Synthetic peptide microarrays provide a rapid, high-throughput platform for immunological testing of potential B-cell epitopes. However, their current capacity prevent the experimental screening of complete "peptidomes". Therefore, computational approaches for prediction and/or prioritization of diagnostically relevant peptides are required. In this work we describe a computational method to assess a defined set of molecular properties for each potential diagnostic target in a reference genome. Properties such as sub-cellular localization or expression level were evaluated for the whole protein. At a higher resolution (short peptides), we assessed a set of local properties, such as repetitive motifs, disorder (structured vs natively unstructured regions), trans-membrane spans, genetic polymorphisms (conserved vs. divergent regions), predicted B-cell epitopes, and sequence similarity against human proteins and other potential cross-reacting species (e.g. other pathogens endemic in overlapping geographical locations). A scoring function based on these different features was developed, and used to rank all peptides from a large eukaryotic pathogen proteome. We applied this method to the identification of candidate diagnostic peptides in the protozoan Trypanosoma cruzi, the causative agent of Chagas disease. We measured the performance of the method by analyzing the enrichment of validated antigens in the high-scoring top of the ranking. Based on this measure, our integrative method outperformed alternative prioritizations based on individual properties (such as B-cell epitope predictors alone). Using this method we ranked [Formula: see text]10 million 12-mer overlapping peptides derived from the complete T. cruzi proteome. Experimental screening of 190 high-scoring peptides allowed the identification of 37 novel epitopes with diagnostic potential, while none of the low scoring peptides showed significant reactivity. Many of the metrics employed are dependent on standard bioinformatic tools and data, so the method can be easily extended to other pathogen genomes.

trans-Sialidase neutralizing antibody detection in Trypanosoma cruzi-infected domestic reservoirs.

The detection of Trypanosoma cruzi infection in domestic dogs and cats is relevant to evaluating human transmission risks and the effectiveness of insecticide spraying campaigns. However, the serological assays routinely used are associated with cross-reactivity in sera from mammals infected with Leishmania spp. We used a trans-sialidase inhibition assay (TIA) for T. cruzi diagnosis in serum samples from 199 dogs and 57 cats from areas where these types of infections are endemic. TIA is based on the antibody neutralization of recombinant trans-sialidase, an enzyme that is not detected in the coendemic Leishmania species or Trypanosoma rangeli parasites. T. cruzi infection was also evaluated by conventional serology (CS) (indirect immunofluorescence, indirect hemagglutination, enzyme-linked immunosorbent assay, and immunochromatographic dipstick test) and xenodiagnosis. Sera from 30 dogs and 15 cats from areas where these organisms are not endemic and 5 dogs with visceral leishmaniasis were found to be nonreactive by TIA and CS. Samples from dogs and cats demonstrated 91 and 95% copositivities between TIA and CS, whereas the conegativities were 98 and 97%, respectively. Sera from xenodiagnosis-positive dogs and cats also reacted by TIA (copositivities of 97 and 83%, respectively). TIA was reactive in three CS-negative samples and was able to resolve results in two cat serum samples that were CS inconclusive. Our study is the first to describe the development of trans-sialidase neutralizing antibodies in naturally infected dogs and cats. High CS conegativity and the absence of trans-sialidase neutralization in dog sera from areas where leishmaniasis is not endemic and from dogs with visceral leishmaniasis support TIA specificity. The TIA may be a useful tool for T. cruzi detection in the main domestic reservoirs.

Immunological identification of Trypanosoma cruzi lineages in human infection along the endemic area.

Genotyping studies show a polarized geographic distribution of Trypanosoma cruzi lineages in humans. Here, we assessed their distribution along Latin America through an immunological approach we designated Western blot (WB) assay with Trypomastigote small-surface antigen (TSSA) I and TSSA II (TSSA-WB). These antigens are expressed by T. cruzi I (TCI; now TcI) and T. cruzi II (TCII; reclassified as TcII to TcVI) parasites. TSSA-WB showed good concordance with genotyping tests. An unexpected frequency of TSSA II recognition was observed in Colombia, Venezuela, and Mexico (northern region of Latin America). In Argentina and Paraguay (southern region), immunophenotyping confirmed the already reported TCII (TcII to TcVI) dominance. The lineage distribution between these regions showed significant difference but not among countries within them (except for Colombia and Venezuela). TSSA-WB shows TCII emergence in the northern region where TCI was reported as dominant or even as the unique T. cruzi lineage infecting humans.

Continuous nonradioactive method for screening trypanosomal trans-sialidase activity and its inhibitors.

Trypanosoma cruzi, the agent of American trypanosomiasis is unable to synthesize sialic acid (SA). Instead of using the corresponding nucleotide sugar as donor of the monosaccharide, the transfer occurs from alpha-2,3-linked SA in the host sialoglycoconjugates to terminal beta-galactopyranosyl units of the parasite mucins. For that purpose, T. cruzi expresses a glycosylphosphatidylinositol-anchored trans-sialidase (TcTS) that is shed into the milieu, being detected in the blood during the acute phase of the infection. The essential role of TcTS in infection and the absence of a similar activity in mammals make this enzyme an attractive target for the development of alternative chemotherapies. However, there is no effective inhibitor toward this enzyme. In vitro, 3'-sialyllactose (SL) as donor and radioactive lactose as acceptor substrate are widely used to measure TcTS activity. The radioactive sialylated product is then isolated by anion exchange chromatography and measured. Here we describe a new nonradioactive assay using SL or fetuin as donor and benzyl beta-d-Fuc-(1-->6)-alpha-d-GlcNAc (1) as acceptor. Disaccharide 1 was easily synthesized by regioselective glycosylation of benzyl alpha-d-GlcNAc with tetra-O-benzoyl-d-fucose followed by debenzoylation. Compound 1 lacks the hydroxyl group at C-6 of the acceptor galactose and therefore is not a substrate for galactose oxidase. Our method relies on the specific quantification of terminal galactose produced by trans-sialylation from the donor to the 6-deoxy-galactose (D-Fuc) unit of 1 by a spectrophotometric galactose oxidase assay. This method may also discriminate sialidase and trans-sialylation activities by running the assay in the absence of acceptor 1.

Estimation of sensitivity and specificity of several Trypanosoma cruzi antibody assays in blood donors in Argentina.

BACKGROUND: The absence of a gold standard test for Trypanosoma cruzi antibodies represents a problem not only for the evaluation of screening tests, but also for appropriate blood donor counseling. The aim of this study was to estimate the sensitivity and specificity of multiple blood donor screening tests for T. cruzi antibodies in Argentina. STUDY DESIGN AND METHODS: From June 2006 to March 2007 a sample of 1455 blood donors was recruited from two blood banks in Chaco province, an area of Argentina with highly endemic T. cruzi infection. Samples were tested by three epimastigote lysate enzyme immunoassays (EIAs), one recombinant antigen EIA, two indirect hemagglutination assay (IHA) tests, a particle agglutination assay (PA), and a research trans-sialidase inhibition assay (TIA). Sensitivity and specificity were estimated using latent class analysis (LCA). RESULTS: LCA estimated the consensus prevalence of T. cruzi infection at 24.5%. Interassay correlation was higher among the four EIA tests and TIA compared to IHA tests. Assay sensitivities varied from 96 to 99.7 for different EIAs, 91% for TIA, 84% for PA, and 66 to 74% for IHA tests. Relative to the LCA, assay specificities were from 96% to almost 100%. CONCLUSION: Based on the comparison of several tests in a large population from an endemic area for T. cruzi infection, our data showed an adequate sensitivity for EIA tests in contrast to PA and IHA assays. The latter tests should no longer be used for blood donor screening.