PCR assay for monitoring Trypanosoma cruzi parasitemia in childhood after specific chemotherapy

Abstract

Assessment of cure of Trypanosoma cruzi infection by antibody seroconversion usually involves several years of follow-up. Parasitological negativity is useless for cure assessment, since even untreated patients mostly show negative results; conversely, positive tests are of great value because they indicate treatment failure. Here, PCR was used to assess the rate of specific chemotherapy failure in a well-characterized Brazilian cohort of T. cruzi-seropositive children, who were enrolled in a field trial of benznidazole (Bz) efficacy. Paired blood samples from 111 children were taken at baseline and 36 months after treatment with either Bz (n = 58) or a placebo (n = 53). DNA extraction and PCR amplification were carried out as previously described, and hybridization was performed with all PCR products. At the end of follow-up, PCR was positive for 39.6% of the patients in the Bz group versus 64.2% in the placebo group (P = 0.01). Untreated patients had a 1.6-fold-higher chance of remaining positive by PCR than those in the Bz group (P < 0.05). We conclude that PCR is a useful tool for revealing therapeutic failure of T. cruzi infection on a short-term basis.

Figures

FIG. 1.

Representative polyacrylamide gel electrophoresis results for blood samples from the Pl and Bz groups of chagasic patients at baseline and 3 years posttreatment analyzed by PCR. (A) MW, molecular weight marker (100-bp ladder; Promega); lane 1, DNA extracted from the blood of a nonchagasic individual (negative control); lanes 2, 4, 6, 8, 10, 12, 14, and 16, DNA from the blood of the Pl group at baseline; lanes 3, 5, 7, 9, 11, 13, 15, and 17, DNA from the blood of the Pl group 3 years later; lanes 18, 20, 22, 24, 26, 28, 30, and 32, DNA from the blood of the Bz group at baseline; lanes 19, 21, 23, 25, 27, 29, 31, and 33, DNA from the blood of the Bz group 3 years posttreatment; lane 34, DNA from the blood of a chronic chagasic patient (positive control); lane 35, no DNA in the reaction mixture for PCR amplification. Arrow indicates T. cruzi-specific products of 330 bp. (B) Slot blot hybridization of PCR products of blood samples from the same individuals.

FIG. 2.

ROC curve of IIF titers, IHA titers, and ELISA index for the identification of children treated with Bz after 3 years of follow-up.