Flow cytometry, a new approach to detect anti-live trypomastigote antibodies and monitor the efficacy of specific treatment in human Chagas' disease

Abstract

Sera from patients chronically infected with Trypanosoma cruzi display antibodies that bind to epitopes of living trypomastigotes, known as lytic antibodies (LA), and are detected by a complement-mediated lysis test. Conventional serology antibodies (CSA) are also present in sera from patients with chronic infections but, in contrast to LA, are unable to recognize viable trypomastigotes. The presence of LA has been used as an important element in the criterion of cure in human Chagas' disease. Using flow cytometry technology, we introduced a new and sensitive immunomethod for the detection of anti-live trypomastigote membrane-bound antibodies. On the basis of serological tests (LA and CSA detection) and parasitological assays such as hemoculture (HE), patients were classified into the following groups: chronically infected untreated patients (NT) and treated not-cured patients (TNC), with positive HE and both LA and CSA in their sera; ''dissociated'' HE-negative patients (DIS), in whom LA was not detected whereas CSA were present; a group of cured HE-negative patients (CUR), who were both LA and CSA negative; and, as control, a group of non-chagasic individuals (NC). Sera from these patients were assayed by incubation with live bloodstream trypomastigotes, which were subsequently exposed to fluorescein isothiocyanate-conjugated anti-human immunoglobulin G. The parasites were then fixed, run in the cytometer, and identified on basis of their size and granularity gain adjustments. On the basis of experience with the complement-mediated lysis test, a level of 20% of parasites being fluorescein isothiocyanate fluorescence positive was used as a cutoff between effective and ineffective treatments. With this criterion, our results indicated that sera from NT and TNC patients were antibody positive whereas all sera from DIS, CUR, and NC patients did not contain membrane-bound antibodies. This new approach is a tool to easily identify anti-live T. cruzi membrane-bound antibodies that can be used to monitor the efficacy of Chagas' disease treatment.

Use of the eie‐recombinant‐chagas‐biomanguinhos kit to monitor cure of human chagas’ disease

“…In a previous study, a new approach using flow cytometry to detect anti-live trypomastigote antibodies and monitor the efficacy of specific treatment in human Chagas' disease was shown to be more sensitive and accurate than CoML (8). However, that method requires expensive equipment (FACScan) which is not commonly available in laboratories.…”

“…Several technical serological approaches using conventional ELISA with purified (6) and recombinant antigens (7), or using flow cytometry technology (8) instead of the CoML test have been evaluated.…”

Influence of parasite presence on the immunologic profile of peripheral blood mononuclear cells from chagasic patients after specific drug therapy

“…Krettli & Brener (1982) have described the presence of antibodies present in chronic chagasic hosts which are able to bind living blood forms of T. cruzi and are associated with active ongoing infection. These anti-live parasite antibodies can be detected and titrated by complement mediated lysis (CoML) (Krettli & Brener 1982), as well as flow cytometric analysis (Martins-Filho et al 1995). A recent study covering a ten year follow up of 82 treated chagasic patients indicated that parasitological cure is associated with both negative results from exhaustive serial haemocultures and absence of anti-live parasite antibodies in patients' blood (Galvão et al 1993).…”

More than a Hundred Years in the Search for an Accurate Diagnosis for Chagas Disease: Current Panorama and Expectations

“…Even with optimized serological assays that use parasite-specific recombinant antigens, inconclusive test results continue to be a problem [58]. However, this technique is utilized for monitoring treatments rather than for diagnosis [59]. In the past decade, several technologies have emerged as diagnostic tools capable of improving diagnosis by using several antigens.…”