Cell Culture and Maintenance of the Evolutionary Forms of <em>Trypanosoma cruzi</em> for Studies of Parasitic Biology

This chapter aims to present and discuss the main cell culture techniques used for the growth and maintenance of the different evolutionary forms of the protozoan Trypanosoma cruzi, the etiologic agent of the Chagas disease. Chagas disease is a neglected tropical disease endemic in several Latin American countries. Here, we intend to present the main difficulties, advantages, and disadvantages of using Trypanosoma cruzi cell culture in parasitic biology. Finally, we present the main research opportunities in the context of Trypanosoma cruzi parasitic biology using cell culture techniques, such as drug development, characterization of pharmacological targets, molecular markers for diagnosis, structural biology, and many other biomedical applications

Identificação e caracterização imunoquímica de antigénios de tripanosomatídeos envolvidos na reatividade serológica entre Trypanosoma cruzi e Leishmania spp.

As doenças tropicais negligenciadas, doença de Chagas e Leishmaniose Visceral, cujos agentes etiológicos são os protozoários Trypanosoma cruzi e Leishmania infantum, respectivamente, exibem distribuição geográfica sobreponível e endemicidades semelhantes em diversas áreas. Trypanosoma cruzi e Leishmania infantum pertencem à mesma família e compartilham repertórios de epítopos antigênicos com proteínas conservadas que podem ser responsáveis pela reatividade serológica cruzada no imunodiagnóstico da doença de Chagas e Leishmaniose Visceral. Reações serológicas cruzadas provocam alterações na precisão do imunodiagnóstico laboratorial destas doenças, podendo gerar distorções em inquéritos serológicos e estudos epidemiológicos. Este estudo realizou a caracterização antigénica e serológica cruzada em soros de indivíduos diagnosticados com a doença de Chagas e Leishmaniose Visceral. Por ELISA foi avaliada a seroreatividade antigénica de Trypanosoma cruzi e Leishmania infantum com a determinação do perfil serológico de anticorpos IgG totais anti Trypanosoma cruzi e anti-Leishmania infantum em amostras de soros de indivíduos com a doença de Chagas (n=240) ou Leishmaniose Visceral (n=240), respectivamente. A pesquisa da reatividade serológica cruzada foi feita por ELISA, quantificando anticorpos IgG totais anti-Trypanosoma cruzi em soros de Leishmaniose Visceral (n=240) e anticorpos IgG totais anti-Leishmania infantum em soros de doença de Chagas (n=240). Utilizou-se a técnica de immunoblotting para detetar frações proteicas de Trypanosoma cruzi e Leishmania infantum em soros de Leishmaniose Visceral e doença de Chagas, respectivamente. Anticorpos IgG totais anti-Trypanosoma cruzi foram detetados em 95,8% (230/240) de soros com doença de Chagas e anticorpos totais anti-Leishmania infantum foram detetados em 94,6% (227/240) de soros com Leishmaniose Visceral. Anticorpos IgG totais anti-Leishmania infantum foram detetados em 91,2% (219/240) de soros com doença de Chagas e anticorpos IgG totais anti-Trypanosoma cruzi foram detetados em 91,2% (219/240) de soros com Leishmaniose Visceral. Conclui-se que anticorpos anti-Trypanosoma cruzi reconhecem o antigénio Leishmania infantum tanto quanto anticorpos anti-Leishmania infantum reconhecem o antigénio Trypanosoma cruzi, em soros de indivíduos infetados com doença de Chagas e Leishmaniose Visceral. A pesquisa pela deteção de proteínas comuns aos parasitas analisados pode ser a diretriz para se chegar a um teste diagnóstico com a sensibilidade e especificidade desejadas e que não provoque seroreatividade cruzada entre o diagnóstico de doença de Chagas e Leishmaniose VisceralNeglected tropical diseases, Chagas disease and Visceral Leishmaniasis, caused by protozoa Trypanosoma cruzi and Leishmania infantum parasites respectively, exhibit geographic distribution overlapping and endemicity. Besides, both protozoans belong to the trypanosomatidae family. Consequently, antigenic epitopes repertoire, manly common conserved proteins, is believed to be responsible for the serological cross reactivity between Chagas disease and Visceral Leishmaniasis. The immunodiagnostics’ precision tests are changed by cross serological reactions. Thus, lead to distortions in serological surveys and epidemiological data analysis. This study carried out the antigenic and serological cross characterization in individuals’ sera diagnosed with Chagas disease and Visceral Leishmaniasis. ELISA assay, the antigenic seroreactivity was used to determine the serological profile of total IgG antibodies anti-Trypanosoma cruzi and anti-Leishmania infantum in serum samples from individuals with Chagas disease (n = 240) or Visceral Leishmaniasis (n = 240). The investigation of serological cross-reactivity was performed also by ELISA, quantifying total IgG antibodies anti Trypanosoma cruzi in individuals’ serums of Visceral Leishmaniasis (n = 240) and total IgG antibodies anti-Leishmania infantum in sera form Chagas disease individuals (n = 240). The immunoblotting technique was used to detect protein fractions of Trypanosoma cruzi and Leishmania infantum in sera of Visceral Leishmaniasis and Chagas disease, respectively. As result, total anti-Trypanosoma cruzi IgG antibodies were detected in 95.8% (230/240) of sera with Chagas disease and total anti-Leishmania infantum antibodies were detected in 94.6% (227/240) of sera with Visceral Leishmaniasis. The cross serological showed that anti-Leishmania infantum IgG antibodies were detected in 91.2% (219/240) of sera with Chagas disease and total anti Trypanosoma cruzi IgG antibodies were detected in 91.2% (219/240) of sera with visceral leishmaniasis. This study concluded that anti-Trypanosoma cruzi antibodies recognize the Leishmania infantum antigen, as well as anti-Leishmania infantum antibodies, recognize the Trypanosoma cruzi antigen, in sera from individuals infected with Chagas disease and Visceral Leishmaniasis. The identification of conserved proteins parasites implicated in cross-reactivity may be the guideline for the improvement of diagnostic tests. Therefore, desirable sensitivity and specificity and that do not cause cross-reactivity between the diagnosis of Chagas disease and Visceral Leishmaniasis

Evaluation of a quality improvement intervention to reduce anastomotic leak following right colectomy (EAGLE): pragmatic, batched stepped-wedge, cluster-randomized trial in 64 countries

Background Anastomotic leak affects 8 per cent of patients after right colectomy with a 10-fold increased risk of postoperative death. The EAGLE study aimed to develop and test whether an international, standardized quality improvement intervention could reduce anastomotic leaks. Methods The internationally intended protocol, iteratively co-developed by a multistage Delphi process, comprised an online educational module introducing risk stratification, an intraoperative checklist, and harmonized surgical techniques. Clusters (hospital teams) were randomized to one of three arms with varied sequences of intervention/data collection by a derived stepped-wedge batch design (at least 18 hospital teams per batch). Patients were blinded to the study allocation. Low- and middle-income country enrolment was encouraged. The primary outcome (assessed by intention to treat) was anastomotic leak rate, and subgroup analyses by module completion (at least 80 per cent of surgeons, high engagement; less than 50 per cent, low engagement) were preplanned. Results A total 355 hospital teams registered, with 332 from 64 countries (39.2 per cent low and middle income) included in the final analysis. The online modules were completed by half of the surgeons (2143 of 4411). The primary analysis included 3039 of the 3268 patients recruited (206 patients had no anastomosis and 23 were lost to follow-up), with anastomotic leaks arising before and after the intervention in 10.1 and 9.6 per cent respectively (adjusted OR 0.87, 95 per cent c.i. 0.59 to 1.30; P = 0.498). The proportion of surgeons completing the educational modules was an influence: the leak rate decreased from 12.2 per cent (61 of 500) before intervention to 5.1 per cent (24 of 473) after intervention in high-engagement centres (adjusted OR 0.36, 0.20 to 0.64; P &lt; 0.001), but this was not observed in low-engagement hospitals (8.3 per cent (59 of 714) and 13.8 per cent (61 of 443) respectively; adjusted OR 2.09, 1.31 to 3.31). Conclusion Completion of globally available digital training by engaged teams can alter anastomotic leak rates. Registration number: NCT04270721 (http://www.clinicaltrials.gov)