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.