Induction of resistance to azole drugs in Trypanosoma cruzi

Abstract

Trypanosoma cruzi is the protozoan parasite that causes Chagas' disease, a frequently fatal illness affecting the heart and gastrointestinal systems. An estimated 16 million to 18 million people in Latin America and 50,000 to 100,000 people in the United States are infected with this pathogen. Treatment options for T. cruzi infections are suboptimal due to the toxicities and limited effectiveness of the available drugs. Azole antimicrobial agents have been discovered to have antitrypanosomal activity by inhibition of ergosterol synthesis. The triazole itraconazole was recently shown to produce a parasitologic cure rate of 53% in chronically infected patients (W. Apt et al., Am. J. Trop. Med. Hyg. 59:133-138, 1998), a result which may lead to more use of this family of drugs for the treatment of T. cruzi infections. In the experiments reported on here, resistance to azoles was induced in vitro by serial passage of mammalian-stage parasites in the presence of fluconazole for 4 months. These parasites were cross resistant to the other azoles, ketoconazole, miconazole, and itraconazole. They remained susceptible to benznidazole and amphotericin B. The azole-resistant phenotype was stable for more than 2 months of in vitro serial passage without fluconazole. In addition, the parasites resisted treatment in mice receiving ketoconazole. The rapid development of azole resistance in T. cruzi in vitro suggests that resistance to azole drugs has the potential to occur in patients and may pose an impediment to the progress being made in the treatment of T. cruzi infection.

FIG. 1

Drug susceptibilities of resistant and parent lines of T. cruzi. Mammalian-stage parasites containing the E. coli β-galactosidase gene were allowed to infect mouse 3T3 fibroblasts in microtiter plates in the presence of the indicated drug dilutions. At day 6, the growth of parasites was quantitated by the addition of the substrate CPRG and reading of the results of the colorimetric reaction at an optical density at 570 nm on an enzyme-linked immunosorbent assay reader. The Fluc^r line of T. cruzi, derived by long-term in vitro passage in the presence of fluconazole, was resistant to four different azole drugs in comparison to the parent line from which it was derived (a to d). There was no cross-resistance to amphotericin B (e) or benznidazole (f).

FIG. 2

Stability of the fluconazole-resistant phenotype. A clone of the Fluc^r parasite line was inoculated into an untreated mouse and was then recovered from blood after 2 weeks. After expansion in vitro (in the absence of fluconazole), the clone (▴) was tested for its susceptibility to fluconazole. It was compared to Fluc^r parasites which were passaged continuously in vitro in the presence of fluconazole (•) and to the parent line of T. cruzi (■). The experiment has been repeated with three separate mice with similar results.

FIG. 3

Infection of mice with azole-resistant T. cruzi. Mice were infected with either the parent clone (1 × 10⁴ trypomastigotes) or the in vitro-derived fluconazole-resistant clone of T. cruzi (2 × 10⁶ trypomastigotes). Mice were treated with either placebo or ketoconazole at 30 mg/kg by oral gavage for the first 7 days of infection (four groups; n = 5 per group). The levels of parasitemia were significantly different between the placebo-treated () and ketoconazole-treated () mice infected with the parent line on all 3 days (P < 0.05), whereas the levels of parasitemia were not significantly different between the placebo-treated (▩) and ketoconazole-treated (■) mice infected with the azole-resistant T. cruzi strain on all 3 days (P > 0.10). hpf, high-power field.