Trypanosoma cruzi Secreted Cyclophilin Tc CyP19 as an Early Marker for Trypanocidal Treatment Efficiency

Abstract

Cyclophilins (CyPs) are a family of enzymes involved in protein folding. Trypanosoma cruzi, the causative agent of Chagas disease, has a 19-kDa cyclophilin, TcCyP19, that was found to be secreted in parasite stages of the CL Brener clone and recognized by sera from T. cruzi-infected mice and patients. The levels of specific antibodies against TcCyP19 in T. cruzi-infected mice and subjects before and after drug treatment were measured by an in-house enzyme linked immunosorbent assay (ELISA). Mice in the acute and chronic phase of infection, with successful trypanocidal treatments, showed significantly lower anti-TcCyP19 antibody levels than untreated mice. In children and adults chronically infected with T. cruzi, a significant decrease in the anti-TcCyP19 titers was observed after 12 months of etiological treatment. This decrease was maintained in adult chronic patients followed-up 30-38 months post-treatment. These results encourage further studies on TcCyP19 as an early biomarker of trypanocidal treatment efficiency.

Keywords: Chagas; ELISA; TcCyP19; Trypanosoma cruzi; benznidazole; biomarker; cyclophilin; nifurtimox; parasiticidal treatment.

Figure 1

The T. cruzi recombinant protein, TcCyP19, electrotransferred onto nitrocellulose strips, was recognized by sera from a chronically T. cruzi-infected patient (H+), a chronically T. cruzi-infected mouse (Mc) and an acute T. cruzi-infected mouse (Ma). As negative controls, sera from an uninfected human (H−) and an uninfected mouse (M−) were used. Polyclonal mouse antibodies against the TcCyP19 recombinant protein were used as a positive control (P).

Figure 2

Anti-TcCyP19 specific antibody levels tested in an in-house ELISA. (A) Serum samples were obtained from C3H/HeN mice infected with a T. cruzi Nicaragua isolate (TcN) treated in the acute phase of the infection with a formulation of Benznidazole in nanoparticles. BNZnp10: mice treated with 10 mg BNZ/kg/day (▲); BNZnp50: mice treated with 50 mg BNZ/kg/day (■) (as described in Section 4.4) Sera from untreated T. cruzi-infected mice (●) were used as a positive control (** p < 0.01). (B) Serum samples were obtained from C57BL/6J mice infected with TcN treated in the chronic phase of the infection. Mice were treated with continuous doses of BNZc75+ALLO (□) and BNZc50+ALLO (ο) or intermittent treatments with one dose of BNZit75+ALLO (♦) or BNZit75 (■) (as described in Section 4.5). Sera from untreated T. cruzi-infected mice were used as positive control (△) (** p < 0.01, * p < 0.05). Results are expressed using the signal-to-cutoff (S/Co) ratio, by dividing the OD value of the samples tested by the OD value of the assay cut-off. The dash line represents S/Co = 1, the ratio obtained with sera from uninfected mice.

Figure 3

Comparison of samples from adult treated patients withdrawn before and after parasiticidal treatment. Antibody levels against TcCyP19 recombinant protein detected in sera from treated patients significantly decreased after treatment. Blood samples after treatment were obtained at a mean of 11.9 months. A p value of 0.0005 (***) indicates a significant difference between the two sample groups using a parametric analysis. The dash line represents S/Co = 1, a ratio obtained with sera from uninfected patients.

Figure 4

Anti-TcCyP19 antibodies - - -■- - - and anti-T. cruzi antibodies -●- by conventional ELISA serology from patients treated with trypanocidal drugs as described in Section 4.8. Post-treatment blood samples obtained from Patient 1 at 4, 26 and 38 months; from Patient 2 at 6, 12, 24 and 37 months; from Patient 3 at 6, 18 and 30 months; and from Patient 4 at 6, 28 and 36 months. A decrease in the reactivity of this recombinant protein can be seen regarding the baseline reaction at 4–6 months after therapy. Results are expressed as OD value at 490 nm.

Figure 5

Anti-TcCyP19 antibodies from children treated with trypanocidal drugs as described in Material and Methods. Post-treatment blood samples were withdrawn at 6, 12 and a mean of 74 months (range 48–120 months). (A): Group of children who achieved seroconversion by conventional serology after treatment. (B) Group of children who sustained serological responses after a 5 year post-treatment follow-up. * p < 0.05.