TESA-blot for the diagnosis of Chagas disease in dogs from co-endemic regions for Trypanosoma cruzi, Trypanosoma evansi and Leishmania chagasi

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Inconclusive results in conventional serological screening for Chagas` disease in blood banks: evaluation of cellular and humoral response

To find the most reliable screening method for Trypanosoma cruzi infection in blood banks. Epidemiological data, lymphoproliferation assay, parasitological, conventional serological tests: immunofluorescence, haemagglutination, ELISA with epimastigote and trypomastigote antigens and reference serological tests: trypomastigote excreted-secreted antigens (TESA) blot and chemiluminescent ELISA assay with mucine from trypomastigote forms were applied to individuals with inconclusive serology, non-chagasic individuals and chronic chagasic patients. TESA blot had the best performance when used as a single test in all the groups. In the inconclusive group 20.5% of individuals were positive for TESA blot, 23.3% for either lymphoproliferation or TESA blot, and 17.8% for lymphoproliferation only. Positive lymphoproliferation without detectable antibodies was observed in 5.47% of all inconclusive serology cases. Analysis of six parameters (three serological assays, at least one parasitological test, one lymphoproliferation assay and epidemiological data) in the inconclusive group showed that diagnosis of Chagas` disease was probable in 15 patients who were positive by two or more serological tests or for whom three of those six parameters were positive. TESA blot is a good confirmatory test for Chagas` disease in the inconclusive group. Although lymphoproliferation suggests the diagnosis of Chagas` disease in the absence of antibodies when associated with a high epidemiological risk of acquiring Chagas` disease, the data from this study and the characteristics of the lymphoproliferation assay (which is both laborious and time-consuming) do not support its use as a confirmatory test in blood-bank screening. However, our findings underscore the need to develop alternative methods that are not based on antibody detection to improve the diagnosis when serological tests are inconclusive.

Aspectos clínico-laboratoriais da infecção natural por Trypanosoma cruzi em cães de Mato Grosso do Sul

A doença de Chagas causada pelo T. cruzi é transmitida, principalmente, por insetos vetores e está distribuída na Argentina, no Chile, na Venezuela e no Brasil. O cão, além de ser um importante reservatório, também é vítima da doença e a única espécie capaz de desenvolver manifestações clínicas iguais a do homem. O presente trabalho teve como objetivo descrever as alterações clínicas encontradas em quatro cães infectados naturalmente pelo T. cruzi e alertar para a possibilidade de que a ocorrência dessa enfermidade em cães de Mato Grosso do Sul possa estar sendo subestimada. Os animais foram selecionados a partir de exames sorológicos de reação de imunofluorescência indireta (RIFI), ensaio imunossorvente ligado à enzima (ELISA) e immunoblotting com antígeno secretado e excretado da forma tripomastigota do T. cruzi (TESA-blot) e submetidos a xenodiagnóstico, exame físico, radiografia torácica, eletrocardiografia, ecocardiografia e bioquímica sérica. As alterações encontradas foram aumento de ventrículo direito, presença de arritmias do tipo bloqueio átrio ventricular, sinus arrest e bloqueio de ramo direito, além de disfunção sistólica e diastólica. Três animais apresentaram hiperproteinemia e as dosagens das enzimas CK e CK-MB revelaram valores indicativos de uma miocardite ativa. Esses são os primeiros casos descritos de cães com evidências consistentes de infecção natural pelo T. cruzi em Mato Grosso do Sul e ressalta-se o alerta aos médicos veterinários para a importância clínica e o papel dessa espécie como reservatório da doença.

Miocardite chagásica em caninos no Estado do Rio Grande do Sul

Neste trabalho, são relatados dois casos de morte súbita por doença de Chagas aguda em caninos da zona rural de Porto Alegre, Rio Grande do Sul, Brasil. Os cães, um macho Pit Bull com nove meses (canino 1) e uma fêmea Labrador Retriever com dois anos (canino 2), morreram em janeiro de 2005 e maio de 2008, respectivamente. As necropsias revelaram aumento cardíaco em ambos os casos. O coração do canino 2 apresentou formato globoso com múltiplas áreas pálidas na musculatura cardíaca, mais evidentes no ventrículo direito e câmaras cardíacas dilatadas, principalmente as da direita. Ao exame histológico, ambos os casos apresentaram alterações semelhantes caracterizadas por infiltrado inflamatório difuso não-purulento acentuado, predominantemente linfocitário intersticial. Nas fibras miocárdicas, havia grande número de pseudocistos, repletos de formas amastigotas do Trypanosoma cruzi. Ao teste sorológico TESA-blot, amostra do canino 2 foi positiva para anticorpos IgM e IgG anti-T.cruzi, achado característico da fase aguda da miocardite chagásica. Os resultados indicam que a doença de Chagas deve ser investigada em casos de morte súbita em cães na região Sul do Brasil e que a espécie pode servir como reservatório e sentinela da doença em humanos.

CLINICAL STUDY of NATURAL INFECTION BY Trypanosoma cruzi IN DOGS RESIDING IN A RURAL AREA IN MATO GROSSO do SUL STATE, BRAZIL

This study was carried out to describe the clinical characteristics of natural infection caused by Trypanosoma cruzi in dogs that reside in a rural area of Mato Grosso do Sul State, Brazil. Conventional and nonconventional diagnostic methods were used for screening T. cruzi infection in 75 dogs that lived in the area. Cardiovascular tests and biochemical examination of sera were also performed in four confirmed positive dogs. The following techniques were employed: indirect immunofluorescence test (IFAT), enzyme-linked immunosorbent assay with T. cruzi epimastigote antigens (EAE-ELISA) and enzyme-linked immunosorbent assay with T. cruzi excreted-secreted trypomastigote antigens (TESA-ELISA) with antibodies detected in 45.33% (n = 34), 24.0% (n = 18) and 12.0% (n = 9) of the dogs, respectively. The current prevalence of the infection was confirmed as 10.7% (n = 8) by immunoblotting test with T. cruzi excreted-secreted antigens (TESA-blot). The test that showed the best concordance index (Kappa; 0.93), sensitivity (100%) and specificity (98.5%) was TESA-ELISA, that when associated with IFAT had the same results as those obtained by TESA-blot (10.7%). Three out of the four chagasic animals showed enlarged cardiac silhouette on X-ray and an increase of the P-wave duration and QRS complex in electrocardiogram. Two dogs presented conduction disturbances, right bundle branch block in one dog and first-degree atrioventricular block and sinus arrest in another. The ecodopplercardiography presented left-ventricular-wall thickness increased during diastole, decrease of the shortening fraction and inversion in the speed peaks of the E and A waves, indicating the presence of systolic and diastolic disorders. The four animals showed enzymatic activities of creatine kinase (221-404 U/L), MB fraction of creatine kinase (189-304 U/L), elevated total proteins (7.6-10.2 g/dL) and total globulins (4.6-7.7g/dL) and reduction of albumin/globulin ratio, which suggested a myocardial injury and continuous antigenic stimulus.

Estudo clínico da infecção natural por T. cruzi em cães residentes em uma área rural de Mato Grosso do Sul, Brasil

Pós-graduação em Medicina Veterinária - FCAV

Trojan Horse or Rorschach Blot? Creative industries discourse around the world

One of the most wide-ranging and sophisticated critiques of creative industries policy argues that it is a kind of Trojan horse, secreting the intellectual heritage of the information society and its technocratic baggage into the realm of cultural practice, suborning the latter's proper claims on the public purse and self-understanding, and aligning it with inappropriate bedfellows such as business services, telecommunications and calls for increases in generic creativity. Reviewing the broad adoption of the concept in policy discourse around the world, this paper suggests that rather than a Trojan horse, it might be better thought of as a Rorschach blot, being invested in for varying reasons and with varying emphases and outcomes. Based on spatial analysis, then, the critique may need modification. Temporally as well, the critique may have been overtaken by later developments taking policy emphases 'beyond' the creative industries.

Evaluation of ELISA coupled with Western blot as a surveillance tool for Trichinella infection in wild boar (Sus scrofa)

Trichinella surveillance in wildlife relies on muscle digestion of large samples which are logistically difficult to store and transport in remote and tropical regions as well as labour-intensive to process. Serological methods such as enzyme-linked immunosorbent assays (ELISAs) offer rapid, cost-effective alternatives for surveillance but should be paired with additional tests because of the high false-positive rates encountered in wildlife. We investigated the utility of ELISAs coupled with Western blot (WB) in providing evidence of Trichinella exposure or infection in wild boar. Serum samples were collected from 673 wild boar from a high- and low-risk region for Trichinella introduction within mainland Australia, which is considered Trichinella-free. Sera were examined using both an 'in-house' and a commercially available indirect-ELISA that used excretory secretory (E/S) antigens. Cut-off values for positive results were determined using sera from the low-risk population. All wild boar from the high-risk region (352) and 139/321 (43.3%) of the wild boar from the low-risk region were tested by artificial digestion. Testing by Western blot using E/S antigens, and a Trichinella-specific real-time PCR was also carried out on all ELISA-positive samples. The two ELISAs correctly classified all positive controls as well as one naturally infected wild boar from Gabba Island in the Torres Strait. In both the high- and low-risk populations, the ELISA results showed substantial agreement (k-value = 0.66) that increased to very good (k-value = 0.82) when WB-positive only samples were compared. The results of testing sera collected from the Australian mainland showed the Trichinella seroprevalence was 3.5% (95% C.I. 0.0-8.0) and 2.3% (95% C.I. 0.0-5.6) using the in-house and commercial ELISA coupled with WB respectively. These estimates were significantly higher (P < 0.05) than the artificial digestion estimate of 0.0% (95% C.I. 0.0-1.1). Real-time PCR testing of muscle from seropositive animals did not detect Trichinella DNA in any mainland animals, but did reveal the presence of a second larvae-positive wild boar on Gabba Island, supporting its utility as an alternative, highly sensitive method in muscle examination. The serology results suggest Australian wildlife may have been exposed to Trichinella parasites. However, because of the possibility of non-specific reactions with other parasitic infections, more work using well-defined cohorts of positive and negative samples is required. Even if the specificity of the ELISAs is proven to be low, their ability to correctly classify the small number of true positive sera in this study indicates utility in screening wild boar populations for reactive sera which can be followed up with additional testing. (C) 2013 Elsevier B.V. All rights reserved.

Active-contour-based automated image quantitation techniques for Western Blot analysis

Western Blot analysis is an analytical technique used in Molecular Biology, Biochemistry, Immunogenetics and other Molecular Biology studies to separate proteins by electrophoresis. The procedure results in images containing nearly rectangular-shaped blots. In this paper, we address the problem of quantitation of the blots using automated image processing techniques. We formulate a special active contour (or snake) called Oblong, which locks on to rectangular shaped objects. Oblongs depend on five free parameters, which is also the minimum number of parameters required for a unique characterization. Unlike many snake formulations, Oblongs do not require explicit gradient computations and therefore the optimization is carried out fast. The performance of Oblongs is assessed on synthesized data and Western Blot Analysis images.

Manejo de la técnica Western Blot. Determinaciones relacionadas con obesidad y diabetes.

23 p.

Ultrasensitive colorimetric detection of protein by aptamer - Au nanoparticles conjugates based on a dot-blot assay

A simple, rapid and ultrasensitive colorimetric detection of protein using aptamer-Au nanoparticles (AuNPs) conjugates based on a dot-blot array has been developed, which was combined with the unique optical properties of AuNPs, enabling the visual detection of protein within minutes without any instrument.

A rapid dot-blot assay to assess chimerism following sex-mismatched bone marrow transplantation

Mixed chimerism may occur more frequently than previously thought following allogeneic bone marrow transplantation and may have implications in terms of relapse, graft-versus-host disease and immune reconstitution. DNA analysis using single or multilocus polymorphic probes cannot reliably discriminate between donor and recipient cells below a level of 10%. We used probe pHY2.1, a cloned segment of tandemly repeated DNA (2000 copies) on the long arm of chromosome Y. A dot blot procedure allowed us to immobilize DNA directly from 50 microliter of peripheral blood or bone marrow. Cross-reactivity was eliminated by hybridization at conditions of extreme stringency (65 degrees C, 50% formamide). Mixing experiments detected male DNA at a level of 0.1% after 10 h exposure. Five patients were studied serially post-bone marrow transplantation. One patient showed mixed chimerism for 12 months, one had complete autologous recovery and the remaining three showed complete engraftment. All results were verified by standard karyotyping on bone marrow cells. This technique is a simple, rapid and sensitive assay for chimerism following sex mismatched bone marrow transplantation.

[Portrait d'homme, à mi-corps, assis, de trois-quarts à droite] : [photographie] / Jean-Baptiste Sabatier-Blot

Ancien possesseur : Gilles, Albert (1873-1959)