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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: 2.01). 02/2007; 318(1-2):102-12. DOI: 10.1016/j.jim.2006.10.009
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ABSTRACT 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.
    Journal of Immunological Methods 07/2014; DOI:10.1016/j.jim.2014.07.005 · 2.01 Impact Factor
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    • "In order to continue this investigation, this study aimed at optimizing the flow cytometry-based method to the diagnosis of Chagas disease patients presenting cardiac disorders after specific chemotherapy. In this work, methodologic changes were incorporated to the original method (Vitelli-Avelar et al., 2007) to increase the sensitivity for the post-therapeutic cure assessment of treated patients. "
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    ABSTRACT: One of the challenges on immunodiagnostic of Chagas disease in endemic areas has been the search for more practical and safe antigenic preparation that provides tests with higher sensitivity and specificity, with low cross-reactivity. A new approach using fixed Trypanosoma cruzi epimastigotes to detect IgG reactivity was investigated previously. In order to continue this investigation, this study aimed at optimizing the flow cytometry-based method to the diagnosis of Chagas disease patients after specific chemotherapy. To achieve our goal, serum samples from 93 subjects - 52 adults chronically infected by T. cruzi, and 41 uninfected controls were tested by flow cytometry. Secondly, serum samples from patients Treated Cured and Treated Uncured from Chagas disease were also tested to evaluate the potential of the method on assessing cure. After establishing the ideal serum dilution and cut off, 121 serum samples from patients with other endemic infections were tested to check cross-reactivity. The results showed that parasite staining with Evan's blue dye eliminated debris, allowing trustworthy analysis of anti-fixed epimastigote IgG reactivity. The applicability of the method to diagnose Chagas disease was confirmed by the high sensitivity (98.1%) and specificity (100%) found. This method also contributed for post-therapeutic assessment of cure, identifying 94.1% of Treated Uncured and 83.3% of Treated Cured patients. Cross-reactivity was observed in a very low number (6.7%). On the whole, these data highly recommend the use of anti-fixed T. cruzi epimastigote IgG reactivity by flow cytometry to the diagnosis and cure monitoring of Chagas disease in endemic areas.
    Journal of immunological methods 06/2011; 369(1-2):22-32. DOI:10.1016/j.jim.2011.03.007 · 2.01 Impact Factor
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    • "During the chronic phase, the direct detection of parasites by xenodiagnosis or hemoculture is difficult and time consuming and the diagnosis is usually done by detecting circulating antibodies with conventional or non-conventional methods (Portela-Lindoso and Shikanai- Yasuda, 2003; Lunardelli et al., 2007). Apart from the usefulness for diagnosis, it has been recently shown that the determination of such antibodies by non-conventional flow cytometry methods is a promising tool to monitor treatment efficacy in Chagas disease patients (Martins-Filho et al., 1995; Martins-Filho et al., 2002; Vitelli-Avelar et al., 2007). Here, we extended our previous studies and compared non-conventional flow cytometry assays and conventional assays (indirect hemagglutination [IHA], indirect immunofluorescence [IIF], enzyme-linked immunosorbent assay [ELISA], and particle gel agglutination [PaGIA]) for their usefulness for prognosis and treatment efficacy in patients 5 years after chemotherapy against Chagas disease. "
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    ABSTRACT: Treatment success of chronically infected Chagas disease patients is laborious and a positive prognosis often is made only after repetitive serological and/or parasitological examinations with continuous negative results. Recently, we have developed a non-conventional flow-cytometric method in order to detect immunoglobulin G antibodies against live trypomastigote forms of Trypanosoma cruzi and showed its usefulness in the prognosis of treatment success. In the present study, we investigated the performance of flow-cytometric anti-live trypomastigote IgG antibodies (FC-ALTA) and flow-cytometric anti-fixed epimastigote IgG antibodies (FC-AFEA), as well as conventional serological methods, for early monitoring of benznidazole treated Chagas disease patients, e.g. 5years after treatment. The analysis of individual FC-ALTA reactivity along the titration curve before and after treatment, we were able to show, that between 4% and 13% of treated patients under evaluation presented with reduced serological reactivity and segregated from the other patient groups. Similar results were obtained with semi-quantitative, conventional indirect hemagglutination or indirect immunofluorescence. Our data therefore suggest that the combined use of conventional and non-conventional serological methods could provide more suitable cure criteria in early post-therapeutic prognosis of Chagas disease.
    Journal of immunological methods 05/2011; 370(1-2):24-34. DOI:10.1016/j.jim.2011.05.003 · 2.01 Impact Factor
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