FLOW-CYTOMETRY, A NEW APPROACH TO DETECT ANTI-LIVE TRYPOMASTIGOTE ANTIBODIES AND MONITOR THE EFFICACY OF SPECIFIC TREATMENT IN HUMAN CHAGAS-DISEASE

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 una ble to recognize viable trypomastigotes, The presence of LA has been u sed as an important element in the criterion of cure in human Chagas' disease. Using flow cytometry technology, we introduced a new and sens itive immunomethod for the detection of anti-live trypomastigote membr ane-bound antibodies. On the basis of serological tests (LA and CSA de tection) and parasitological assays such as hemoculture (HE), patients were classified into the following groups: chronically infected untre ated patients (NT) and treated not-cured patients (TNC), with positive HE and both LA and CSA in their sera; ''dissociated'' HE-negative pat ients (DIS), in whom LA was not detected whereas CSA were present; a g roup of cured HE-negative patients (CUR), who were both LA and CSA neg ative; 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 isothi ocyanate-conjugated anti-human immunoglobulin G. The parasites were th en 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 fluo rescein isothiocyanate fluorescence positive was used as a cutoff betw een effective and ineffective treatments, With this criterion, our res ults indicated that sera from NT and TNC patients were antibody positi ve whereas all sera from DIS, CUR, and NC patients did not contain mem brane-bound antibodies, This new approach is a tool to easily identify anti-live T. cruzi membrane-bound antibodies that can be used to moni tor the efficacy of Chagas' disease treatment.