Double-blind study to evaluate flow cytometry analysis of anti-live trypomastigote antibodies for monitoring treatment efficacy in cases of human Chagas' disease

Abstract

The validation of flow cytometry analysis of anti-live trypomastigote antibodies (FC-ALTA) to monitor cure after treatment of Chagas' disease was evaluated with serum samples from treated and nontreated chagasic patients. After optimization of the original technique, toward better sensitivity and applicability to field surveys, we design a double blind study of 94 coded samples classified into the following categories: patients not treated (NT) and patients treated but not cured (TNC), both presenting positive conventional serology and xenodiagnosis; patients treated and cured (TC), showing negative serology and xenodiagnosis; and patients treated under evaluation (TUE), who presented positive or oscillating conventional serology (CSA) but negative xenodiagnosis. Coded samples, diluted 1:256, were assayed by incubation with live cell culture trypomastigotes, which were subsequently stained with fluorescein isothiocyanate-conjugated anti-human immunoglobulin G, with prior fixation and analysis by flow cytometry. The results were expressed as the percentages of positive fluorescent parasites (PPFP) for each individual sample, establishing 20% PPFP as the cutoff between negative and positive results. Our data demonstrated that all NT and TNC presented positive results while all but one TC had a PPFP lower than 20%. Analysis of TUE demonstrated a wide degree of reactivity, with PPFP values that were negative (PPFP </= 20%), low positive (20% < PPFP </= 50%), and high positive (PPFP > 50%). As TUE with negative PPFP presented negative xenodiagnosis and positive or oscillating CSA, they were classified as dissociated according to the criteria of Krettli and Brener (J. Immunol. 128:2009-2012, 1982) and could indeed be considered cured after chemotherapy. This study demonstrates and validates the use of FC-ALTA to easily identify anti-live trypomastigote membrane-bound antibodies, offering another approach for investigating and monitoring the efficacy of specific chemotherapy in cases of human Chagas' disease.

FIG. 1.

FC-ALTA. (a) Dot plot analysis of a representative trypomastigote distribution (R1) based on size and granularity. Single histograms represent PPFP values for FITC-conjugated anti-human IgG internal control (b), negative control (c), and positive control (d).

FIG. 2.

Representative histograms showing effect of incubation temperature on PPFP values. Parasites were incubated in parallel study with a 1:256 dilution of PBS-10% FBS-diluted human sera (from NT and TC patients) followed by a 1-h incubation at 4°C (a) or a 30-min incubation at 37°C (b) with FITC-conjugated anti-human IgG (whole-molecule) antibody. (c) A range of samples were tested for both protocols (−4°C for 60 min [○] and 37°C for 30 min [•]), including samples from the NT (n = 12), TNC (n = 9), and TC (n = 4) patient groups. Statistical analysis demonstrated significant differences between PPFP values obtained at 37°C for 60 min in comparison to those obtained at 4°C for 60 min for the NT and TNC patient groups but not for the TC patient group.

FIG. 3.

Comparison of PPFP values between pure and GPS. Parallel studies with pairs of FS (•) and GPS (○) samples were carried out, and the results were presented as PPFP values. Samples were grouped based on the PPFP values obtained with FS followed by comparison with the PPFP values obtained with GPS. No significant differences were observed between mean PPFP values obtained from FS and GPS.

FIG. 4.

Analysis of PPFP values from patients with positive xenodiagnosis and positive conventional serology. The performance of FC-ALTA with samples from the NT (a) and TNC (b) patient groups was investigated by using FS, and data analysis was performed by establishing a 20% PPFP value as the cutoff between negative and positive results, as described by Martins-Filho et al. (18). Positive results were additionally classified according to the method of Cordeiro et al. (9) as low positive (20% < PPFP ≤ 50%) and high positive (PPFP > 50%). The TNC patients were subdivided into two groups, as they received treatment during the acute (A) or chronic (C) phase of the disease.

FIG. 5.

Analysis of PPFP values from patients with negative xenodiagnosis and negative conventional serology. The performance of FC-ALTA for samples from the TC patient group was investigated by using FS, and data analysis was performed by establishing a 20% PPFP value as the cutoff between negative and positive results, as described by Martins-Filho et al. (18). Positive results were additionally classified according to the method of Cordeiro et al. (9) as low positive (20% < PPFP ≤ 50%) and high positive (PPFP > 50%). The TC patients were subdivided into three groups, as they received treatment during the acute (A), subacute (SA), or chronic (C) phase of the disease.

FIG. 6.

Analysis of PPFP values from patients with negative xenodiagnosis but positive or oscillating conventional serology. The performance of FC-ALTA for samples from the TUE patient group was investigated by using FS, and data analysis was performed by establishing a 20% PPFP value as the cutoff between negative and positive results, as described by Martins-Filho et al. (18). Positive results were additionally classified according to the method of Cordeiro et al. (9) as low positive (20% < PPFP ≤ 50%) and high positive (PPFP > 50%). The TC patients were subdivided into groups, as they received treatment during the acute (A) or chronic (C) phase of the disease (b), had positive or oscillating serology (a), and had less or more than 10 years of follow-up after treatment (c).