Non-conventional flow cytometry approaches to detect anti-Trypanosoma cruzi immunoglobulin G in the clinical laboratory

Laboratório de Doença de Chagas, Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz, Brazil.
Journal of Immunological Methods (Impact Factor: 1.82). 02/2007; 318(1-2):102-12. DOI: 10.1016/j.jim.2006.10.009
Source: PubMed


We have recently developed a flow cytometric approach to detect anti-live trypomastigote and anti-fixed epimastigote IgG antibodies (FC-ALTA and FC-AFEA) in sera from individuals infected by Trypanosoma cruzi. Here, we present the first evaluation of the applicability of FC-AFEA-IgG as a diagnostic tool for Chagas disease. Performance analysis demonstrated that FC-AFEA-IgG has a sensitivity of 82% and a specificity of 100%. The assessment for prognosis performed by FC-ALTA-IgG1 and FC-AFEA-IgG, after classification of chagasic patients as belonging to indeterminate (IND), cardiac (CARD) or digestive (DIG) clinical forms, showed that most of IND have higher amounts of IgG than individuals' carrying CARD or DIG Chagas disease. FC-AFEA-IgG was also evaluated as a method to monitor chemotherapy efficacy in individuals classified into three distinct categories: not treated (NT), treated but not cured (TNC), and treated and cured (TC). Performance analysis demonstrated that FC-AFEA-IgG has an extraordinary capacity as a serological criterion to assess cure after therapeutic intervention in Chagas disease. These results represent a great advance in the application of serological techniques for clinical investigations on Chagas disease, and they clearly define new directions and perspectives. We intend to continue this field research focusing our attention on the influence of the degree of clinical damage on the FC-ALTA-IgG1 and FC-AFEA-IgG reactivity.

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    • "These findings demonstrated an outstanding performance of FC-ATE for the post-therapeutic monitoring of Chagas disease. The results were consistent with those found for FC-ALTA and FC-AFEA (Martins-Filho et al., 1995, 2002; Vitelli-Avelar et al., 2007; Matos et al., 2011). Considering that the results found in FC-ATE were consistent with those detected for FC-ALTA and FC-AFEA, a comparative analysis of these both techniques with FC-ATE was performed using serum samples from the NT, TNC and TC groups. "
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    ABSTRACT: This study developed a remarkable methodological innovation (FC-ATE) which enables simultaneous detection of antibodies specific to the three evolutive forms of Trypanosoma cruzi: live amastigote (AMA), live trypomastigote (TRYPO), and fixed epimastigote (EPI) using a differential fluorescence staining as low (AMA), intermediate (TRYPO), and high (EPI). An outstanding performance (100%) was observed in the discrimination of chagasic (CH), and non-chagasic (NCH) patients. In the applicability of FC-ATE in the diagnosis of Chagas disease,100% of the CH samples presented positivity in the percentage of positive fluorescent parasites (PPFP) for all three forms of T. cruzi. Moreover, 94% of the samples of NCH presented negatives values of PPFP with AMA and TRYPO, and 88% with EPI. Samples from NCH group with false-positive results were those belonging to the leishmaniasis patients. Considering the applicability of this technique in post-therapeutic monitoring of Chagas disease, 100% of non-treated (NT), and treated non-cured (TNC) samples were positive with the three T. cruzi evolutive forms, while a percentage of 100% from samples of the treated cured (TC) patients were negative with AMA, 93% with TRYPO, and 96% with EPI. The comparison between FC-ATE and two other flow cytometric tests using the same samples of patients NT, TNC and TC showed that the three techniques presented different reactivity, although categorical correlation between the methodologies were observed. Taken together, the results obtained with the novel FC-ATE method have shown an outstanding performance in the diagnosis and post-therapeutic monitoring of Chagas disease.
    Full-text · Article · Jul 2014 · Journal of Immunological Methods
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    • "Research on the discovery and use of new antigens for diagnosis is on-going [42-44], but new diagnostic techniques such as proteomics [36] and flow cytometry [45] are also being investigated. In this study, we looked at the effect of Nfx in Chagas patients by using proteomics on samples from the same cohort of adults described in our previous study. "
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    ABSTRACT: Background Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, remains an important public health issue in many Central and South American countries, as well as non-endemic areas with high rates of immigration from these countries. Existing treatment options for CD are limited and often unsatisfactory. Moreover the lack of post-treatment tests of cure limits the development of new drugs. To address this issue, we sought to identify serum biomarkers following nifurtimox (Nfx) treatment that could be used as an early test of cure and/or markers of a therapeutic response. Methods Human sera from Chagas patients pre- and post-treatment with Nfx (n = 37) were compared to samples from healthy subjects (n = 37) using a range of proteomic and immunologic techniques. Biomarker peaks with the best discriminatory power were further characterized. Results Using serum samples (n = 111), we validated the presence of five key biomarkers identified in our previous study, namely human apolipoprotein A-I (APOA1) and specific fragments thereof and one fragment of human fibronectin (FN1). In chagasic serum samples all biomarkers except full-length APOA1 were upregulated. These five biomarkers returned to normal in 43% (16/37) of the patients treated with Nfx at three years after treatment. Conclusions The normalization of biomarker patterns strongly associated with CD suggests that these markers can be used to identify patients in whom Nfx treatment is successful. We believe that these are the first biomarkers predictive of cure in CD patients.
    Full-text · Article · Jun 2014 · BMC Infectious Diseases
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    • "Labeling of parasites for flow cytometry was performed essentially as described by Vitelli-Avelar et al., with slight modifications [45]. In short, live trypomastigotes (106/assay) were incubated for 1 h at 4°C with anti-TcTASV-C or control antibodies in PBS 10% bovine fetal serum. "
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    ABSTRACT: Among the several multigene families codified by the genome of T. cruzi, the TcTASV family was the latest discovered. The TcTASV (Trypomastigote, Alanine, Serine, Valine) family is composed of ∼40 members, with conserved carboxi- and amino-termini but with a variable central core. According to the length and sequence of the central region the family is split into 3 subfamilies. The TcTASV family is conserved in the genomes of - at least - lineages TcI and TcVI and has no orthologues in other trypanosomatids. In the present work we focus on the study of the TcTASV-C subfamily, composed by 16 genes in the CL Brener strain. We determined that TcTASV-C is preferentially expressed in trypomastigotes, but it is not a major component of the parasite. Both immunoflourescence and flow cytometry experiments indicated that TcTASV-C has a clonal expression, i.e. it is not expressed by all the parasites of a certain population at the same time. We also determined that TcTASV-C is phosphorylated and glycosylated. TASV-C is attached to the parasite surface by a GPI anchor and is shed spontaneously into the medium. About 30% of sera from infected hosts reacted with TcTASV-C, confirming its exposition to the immune system. Its superficial localization and secretory nature suggest a possible role in host-parasite interactions.
    Full-text · Article · Jul 2013 · PLoS ONE
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