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      Estandarización de la técnica de western blot para el diagnóstico específico de la enfermedad de Chagas utilizando antígenos de excreción-secreción de los epimastigotes de Trypanosoma cruzi Translated title: Western blot technique standardization for specific diagnosis of Chagas disease using excretory-secretory antigens of Trypanosoma cruzi epimastigotes

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          Abstract

          Objetivos. Evaluar la eficacia del Western Blot para el diagnóstico específico de la enfermedad de Chagas utilizando antígenos de excreción-secreción de epimastigotes de Trypanosoma cruzi. Materiales y métodos. Los antígenos fueron obtenidos luego de veinte horas de incubación en medio Minimum Essential Medium-Eagle, los cuales fueron preparados a la concentración proteica de 0,2 ug/uL para ser enfrentados a 10 mL de un pool de sueros de pacientes con la enfermedad de Chagas y un conjugado anti-IgG marcado con peroxidasa, se evidenció la presencia de los antígenos de 10, 12, 14,15, 19, 20, 23, 26, 30, 33, 36, 40, 42, 46, 58, 63, 69 91,100 y 112 KDa, de los cuales, los antígenos de 10, 12, 14, 15, 19, 20, 23 y 26 KDa, han sido considerados como específicos usando pools de sueros de pacientes con otras parasitosis y sueros de personas no parasitadas. La sensibilidad de la técnica se evaluó empleando sueros individuales de 65 pacientes con la enfermedad de Chagas y la especificidad con sueros de 40 pacientes con otras parasitosis y cinco sueros de personas no parasitadas. Resultados. La técnica tiene una sensibilidad de 95,4% en la detección de una a ocho bandas específicas, una especificidad de 100%, un valor predictivo positivo de 100% y un valor predictivo negativo de 93,7%. Conclusiones. La técnica de Western Blot con antígenos de excreción-secreción de epimastigotes de T. cruzi es eficaz en el diagnóstico de la enfermedad de Chagas en el Perú, pudiendo ser utilizada como prueba confirmatoria.

          Translated abstract

          Objectives. Evaluate the effectiveness of Western Blot for the specific diagnosis of Chagas disease using excretory-secretory antigens of Trypanosoma cruzi epimastigotes. Materials and methods. Antigens were obtained after twenty hours of incubation in Eagle’s Minimum Essential Medium, which were prepared at a protein concentration of 0.2 ug/uL to be faced with 10 mL pool of serum from patients with Chagas disease and a conjugated anti-IgG labeled with peroxidase. The presence of the following antigens was observed: 10, 12, 14, 15, 19, 20, 23, 26, 30, 33, 36, 40, 42, 46, 58, 63, 69, 91, 100, and 112 kDa; of which antigens of 10, 12, 14, 15, 19, 20, 23, and 26 kDa were considered to be specific using pools of serum from patients with other parasitosis and serum from people with no parasites. The sensitivity of the technique was assessed using individual serum from 65 patients with Chagas disease; and the specificity with serum from 40 patients with other parasitosis, and serums from five people who did not have parasites. Results. The technique has a sensitivity of 95.4% in the detection of one to eight specific bands, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 93.7%. Conclusions. Western Blot technique with excretory-secretory antigens of T. cruzi epimastigotes is effective in the diagnosis of Chagas disease in Peru; therefore, it can be used as a confirmatory test.

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          WHO comparative evaluation of serologic assays for Chagas disease.

          Evaluation of commercially available test kits for Chagas disease for use in blood bank screening is difficult due to a lack of large and well-characterized specimen panels. This study presents a collaborative effort of Latin American blood centers and the World Health Organization (WHO) to establish such a panel. A total of 437 specimens, from 10 countries were collected and sent to the WHO Collaborating Center in São Paulo and used to evaluate 19 screening assays during 2001 through 2005. Specimens were assigned a positive or negative status based on concordant results in at least three of the four confirmatory assays (indirect immunofluorescence, Western blot, radioimmunoprecipitation assay, and recombinant immunoblot). Of the 437 specimens, 168 (39%) were characterized as positive, 262 (61%) were characterized as negative, and 7 (2%) were judged inconclusive and excluded from the analysis. Sensitivity and specificity varied considerably: 88 to 100 and 60 to 100 percent, respectively. Overall, enzyme immunoassays (EIAs) performed better than the other screening assays. Four EIAs had both parameters higher than 99 percent. Of the four confirmatory assays, only the RIPA gave a 100 percent agreement with the final serologic status of the specimens. The sensitivities and specificities of at least four of the commercially available EIAs for Chagas disease are probably high enough to justify their use for single-assay screening of blood donations. Our data suggest that the majority of commercially available indirect hemagglutination assays should not be used for blood donor screening and that the RIPA could be considered a gold standard for evaluating the performance of other assays.
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            Chagas Disease Has Now Gone Global

            Chagas disease, caused by the parasite Trypanosoma cruzi, was once thought to be an exotic disease, confined to endemic areas of Latin America and hence of little importance to anyone outside of these endemic regions, including most physicians and scientists. The impact of the lack of physician awareness and lack of scientific attention is undefined, but may contribute to the continued neglect of Chagas disease and the affected populations. Despite historical evidence and growing recognition of the spread of Chagas disease, the prevention and control of this disease outside of Latin America is only now being addressed. Chagas disease was recognized in the United States as early as the 1950s, when the first reports of local vector-borne cases were published [1]. More recently, immigration patterns from endemic countries have changed the epidemiology of this disease in the US. In 1985, Kirchhoff reported three Bolivian immigrants who presented to the US National Institutes of Health with clinical Chagas disease [2], and in 1987 a survey of Central American immigrants in the Washington, D.C., area revealed a 4.9% prevalence of Chagas disease in this population [3]. Shortly after these reports, cases of transfusion-associated Chagas disease were identified in New York City, US, and Manitoba, Canada [4], [5]. In the New York City case, the donor was traced to a Bolivian immigrant and the recipient was a 12-year-old girl with Hodgkin’s disease. Kirchhoff, in an accompanying editorial, raised the alarm as to whether the blood supply was safe [6]; however, it was not until 15 years later that a screening test for Chagas disease was approved by FDA and implemented by the American blood banking industry. To date, this screening has resulted in the recognition of over 1,300 cases of Chagas disease in donors (http://www.aabb.org/programs/biovigilance/Pages/chagas.aspx), the vast majority of which have been asymptomatic representing the indeterminate form of chronic infection. In other parts of the world, immigration alone has contributed to the appearance of Chagas disease in non-endemic countries [7]–[10]. Immigration from endemic regions is widespread; for example, there are Brazilian immigrants in Portugal and Bolivian immigrants in Spain, and currently, there are an estimated 100,000 or more Latin American immigrants living in France. With immigration has come Chagas disease. Chagasic heart disease has been reported in Brazilian immigrants of Japanese origin in Japan [8], and the seroprevalence of Chagas disease among Bolivian women in Barcelona has been determined to be 3.4% [8]. In all parts of the world where people at risk for Chagas disease are found, Chagas disease in immune-suppressed patients has become an important consideration, resulting in organ and tissue safety concerns related to both donors and recipients. In non-endemic areas, screening of donors or recipients may not be performed routinely. Furthermore, individuals with chronic Chagas disease who acquire HIV/AIDS may have a recrudescence of the infection that can go unrecognized or misdiagnosed as Toxoplasma encephalitis. Most of those infected have the indeterminate, asymptomatic form of Chagas disease and are unaware of their infection, but remain potential sources of transmission. Pregnant women unaware of their infection can be sources of congenital transmission. Congenital Chagas disease has now been reported in Europe among infants born to mothers who are Latin American immigrants with undiagnosed Chagas disease [11]–[13]. These observations raise the issue as to whether prescreening of pregnant women for Chagas disease should be recommended for immigrants from Chagas-endemic areas. This issue was recently highlighted in a paper by Verani et al. [14], who conducted a survey of obstetricians and gynecologists in the US, and demonstrated that clinicians had an inadequate understanding of basic information about this disease and no knowledge of the fact that Chagas disease could be transmitted from mother to child. The paper in this issue of PLoS Neglected Tropical Diseases by Roca et al. [15] examined the prevalence of Chagas disease among Latin American immigrants in a primary care setting in Barcelona, which has become a destination of Spanish-speaking immigrants from Chagas-endemic areas. Of the 766 patients tested, 22 individuals were diagnosed with T. cruzi infection (a prevalence of 2.8%); more women were positive than men (54.6% versus 45.5%). Interestingly, 21 patients were from Bolivia, which is a highly endemic area. The prevalence rate among Bolivian immigrants in this study was 16.5%. Many had lived in substandard adobe houses that have been associated with risk for transmission while in Bolivia, and had previous knowledge of Chagas disease in their country of origin. A number of these patients had clinical Chagas disease, including cardiac and gastrointestinal manifestations. Awareness that Chagas disease is now found in places far from endemic areas of Latin America is important because it leads to the development of strategies to prevent potential sources of transmission (e.g., blood transfusion, organ transplantation, or congenital transmission), and to identify individuals who may benefit from anti-parasitic therapy. Increased awareness also enables us to identify patients who may have a diagnosis of ischemic heart disease or cardiomyopathy of unknown etiology or individuals with gastrointestinal disorders of unknown etiology whose illness is actually Chagas disease, improving the ability of physicians to care for these patients appropriately. Indeed, it is evident that the challenges of Chagas disease have become global.
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              Working to Overcome the Global Impact of Neglected Tropical Diseases: the First WHO Report on Neglected Tropical Diseases

              (2010)
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                Author and article information

                Contributors
                Role: ND
                Role: ND
                Role: ND
                Role: ND
                Role: ND
                Role: ND
                Journal
                rpmesp
                Revista Peruana de Medicina Experimental y Salud Pública
                Rev Peru Med Exp Salud Publica
                Instituto Nacional de Salud (Lima )
                1726-4634
                October 2014
                : 31
                : 4
                : 644-651
                Affiliations
                [1 ] ESCACORP Perú
                [2 ] Universidad Nacional de Trujillo Perú
                Article
                S1726-46342014000400005
                96af3789-6f88-45ed-b213-0718dd6d39e5

                http://creativecommons.org/licenses/by/4.0/

                History
                Categories
                Health Policy & Services

                Public health
                Trypanosoma cruzi,Chagas disease,Western Blot,Parasitic sensitivity tests,Sensitivity and specificity,Enfermedad de Chagas,Pruebas de sensibilidad parasitaria,Sensibilidad y especificidad

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