RESUMO A doença de Chagas (DC) é uma doença tropical crônica causada pelo Trypanosoma cruzi, atingindo cerca de 8 milhões de pessoas na América Latina. A via das lectinas (VL) do sistema complemento é uma das primeiras linhas de defesa na resposta imunológica contra a infecção pelo T. cruzi, e pode continuar sendo ativada na infecção crônicadevido ao escape do parasito à sua ação. Embora alguns componentes dessa via tenham sido investigados na DC, não existem relatos sobre sua ativação em soro de pacientes. Neste contexto, nosso objetivo foi avaliar a ativação da VL no soro de pacientes com DC crônica e controles pela detecção do componente C4 empregando a técnica de ELISA. Para isso, amostras de soro de 80 pacientes com DC crônica (formas clínicas: indeterminada n=17; sintomática n=63; cardíaca n=45; cardiodigestiva n=13; digestiva n=5) atendidos no Ambulatório de Atenção ao Paciente Chagásico (HC/UFPR) e 80 controles saudáveis (doadores do Banco de Sangue do HC) foram avaliados quanto a ativação da VL. Os resultados demonstraram que a ativação da VL pela lectina ligante de manose (MBL) encontra-se reduzida, enquanto que a ativação pelas ficolinas está aumentada em pacientes com DC quando comparados aos controles. Os mesmos resultados foram observados quando os pacientes foram categorizados quanto às formas clínicas indeterminada e sintomática. Concluímos que a detecção do componente C4 por ELISA é uma metodologia eficiente para avaliar a ativação da VL em soro de pacientes com DC crônica possibilitando diferenciar o perfil de ativação entre pacientes e controles.

ABSTRACT Chagas disease (CD) is a chronic tropical disease caused by Trypanosoma cruzi , affecting about 8 million people in Latin America. The lectin pathway (LP) of the complement system is one of the first lines of host defense in the response against T. cruzi , and can continue to be activated in chronic infection due to the escape of the parasite to its action. Although some components of this pathway have been investigated in CD, there are no reports on its activation in patient serum. In this context, our objective was to evaluate the activation of LP in chronic chagasic patients and controls by the detection of the C4 component, using the direct ELISA assay. For this purpose, serum of 80 patient with chronic CD (clinical forms: asymptomatic n=17; symptomatic n=63; cardiac n=45; cardio digestive n=13; digestive n=5) followed at the Ambulatory of Attention to Chagasic Patients (HC/UFPR) and 80 healthy controls (donors of the Blood Bank of HC) were evaluated regarding the evaluation of the LP. The results showed that LP activation by mannose-binding lectin (MBL) was found reduced while activation by ficolins was increased in patients with CD when compared to controls. The same results were observed when the patients were categorized according to the indeterminate and symptomatic clinical forms. We conclude that the detection of the C4 component by ELISA is an efficient methodology to assess LP activation in serum from patients with chronic CD, enabling to differentiate the activation profile between patients and controls..

Currently, treatment of Chagas disease is restricted to the use of benznidazole and nifurtimox; however, nifurtimox was temporarily suspended in some countries. Based on the limitations in pharmacological treatments, it is essential to guarantee the quality of benznidazole. Therefore, this work aimed to identify the degradation products of benznidazole and to validate a stability indicating method. The stability test was performed through a forced degradation study, exposing benznidazole under a range of conditions. Benznidazole proved to be unstable under acidic and alkaline conditions, and three products were identified by liquid chromatography coupled to a hybrid quadrupole time-of-flight mass spectrometer (UPLC-QTOF-MS). Furthermore, a stability indicating method by high performance liquid chromatography with a diode array detector (HPLC-DAD) for benznidazole was developed and validated according to ICH (International Conference on Harmonisation) and ANVISA (Agência Nacional de Vigilância Sanitária) guidelines. The method was linear (180-420 µg mL-1 range), selective in the presence of degradation products, precise (relative standard deviation (RSD) < 0.6%), accurate (recovery > 99.2%), robust, and free of matrix effects. The method was successfully able to quantify the benznidazole in pharmaceutical formulations.