Effects of ravuconazole treatment on parasite load and immune response in dogs experimentally infected with Trypanosoma cruzi

Abstract

In this work, we investigated the in vivo activity of ravuconazole against the Y and Berenice-78 Trypanosoma cruzi strains using acutely infected dogs as hosts. Ravuconazole was well tolerated, as no significant side effects were observed during the treatment using 6.0 mg/kg twice a day (12 mg/kg/day) for up to 90 days. In all treated animals, parasitemia was permanently suppressed by the first day of treatment, independently of the parasite strain. Cultures of blood obtained posttreatment were negative for 90% of the animals, confirming that the drug induced a marked reduction in the parasite load. The results of PCR tests for T. cruzi in blood performed 1 month posttreatment were consistently negative for three of five and two of five animals infected with the Y and Berenice-78 strains, respectively. All ravuconazole-treated dogs consistently had negative serological test results during and until 30 days after treatment, regardless of the therapeutic scheme used. However, after the end of treatment, an increase in specific antibody levels was observed in all treated animals, although the antibody levels were always significantly lower than those of the nontreated control dogs. Despite being unable to induce a parasitological cure, ravuconazole treatment led to significant reductions in the levels of gamma interferon expression and lesions in cardiac tissues in animals infected with the Y strain, while the level of interleukin-10 mRNA expression increased. We conclude that ravuconazole has potent suppressive but not curative activity in the canine model of acute Chagas' disease, probably due to its unfavorable pharmacokinetic properties (half-life, 8.8 h). The longer half-life of ravuconazole in humans (4 to 8 days) makes it a promising drug for assessment for use as chemotherapy in human Chagas' disease.

FIG. 1.

Weight gain (in kilograms) of animals infected with the Y and Berenice-78 Trypanosoma cruzi strains and treated with RAV or BZ compared with the weight gains of the infected untreated (infected control [IC]) and noninfected control (NIC) groups 1 day (A) and 180 days (B) after the end of treatment. The weight gain was calculated from the difference between the weights of the animals assessed on the first and the last days of treatment.

FIG. 2.

Parasitemia curve for dogs inoculated with 2,000 blood trypomastigotes/kg of body weight of the Y (A) and Berenice-78 (B) T. cruzi strains and left untreated.

FIG. 3.

IgG, IgG1, and IgG2 antibody levels in sera of dogs inoculated with 2,000 trypomastigotes per kg of body weight of the T. cruzi Y strain and treated with RAV at 12 mg/kg b.i.d. (q12h) for 90 days or BZ at 7 mg/kg b.i.d. (q12h) for 60 days compared with the levels in the infected and untreated (infected control [IC]) and noninfected control (NIC) groups. T1, 15 days after infection, before treatment; T2, 75 days of infection or 60 days of treatment; T3, 255 (BZ-treated animals) and 285 (RAV-treated animals) days after infection or 180 days posttreatment.

FIG. 4.

IgG, IgG1, and IgG2 antibody levels in the sera of dogs inoculated with 2,000 trypomastigotes per kg of body weight of the Berenice-78 T. cruzi strain and treated with RAV at 12 mg/kg b.i.d. (q12h) for 90 days or BZ at 7 mg/kg b.i.d. (q12h) for 60 days compared with the levels in the infected and untreated (infected control [IC]) and noninfected control (NIC) groups. T1, 15 days after infection or before treatment; T2, 75 days of infection or 60 days of treatment; T3, 255 (BZ-treated animals) and 285 (RAV-treated animals) days of infection or 180 days posttreatment. IgG cutoff, 0.338; IgG1 cutoff, 0.170; IgG2 cutoff, 0.315.

FIG. 5.

Effect of specific treatment on the intensity of lesions or cytokine expression in the heart tissue of dogs infected with Trypanosoma cruzi. Dogs were inoculated with 2,000 trypomastigotes per kg of body weight of the Y strain and treated with RAV at 12 mg/kg b.i.d. (q12h) for 90 days or BZ at 7 mg/kg b.i.d. (q12h) for 60 days; infected and untreated (infected control [IC]) and noninfected control (NIC) groups were also evaluated. IFN-γ (A) and IL-10 (B) mRNA expression in the right atrium was evaluated by semiquantitative reverse transcription-PCR 180 days posttreatment. Morphometric and histopatology analyses were performed with hematoxylin-eosin staining for inflammation quantification (C, E, F, G, and H) and Masson's trichrome staining for collagen quantification (D, I, J, K, and L). Magnifications, ×40. a, b, and c, different letters indicate a significant difference (P < 0.05).

FIG. 6.

Effect of specific treatment on the intensity of lesions or cytokine expression in the heart tissue of dogs infected with Trypanosoma cruzi. Dogs were inoculated with 2,000 trypomastigotes per kg of body weight of the Berenice-78 strain and treated with RAV at 12 mg/kg b.i.d. (q12h) for 90 days or BZ at 7 mg/kg b.i.d. (q12h) for 60 days; infected and untreated controls (infected control [IC]) and noninfected control (NIC) groups were also evaluated. IFN-γ (A) and IL-10 (B) mRNA expression in the right atrium was evaluated by semiquantitative reverse transcription-PCR 180 days posttreatment. Morphometric and histopatology analyses were performed with hematoxylin-eosin staining for inflammation quantification (C, E, F, G, and H) and Masson's trichrome staining for collagen quantification (D, I, J, K, and L). Magnifications, ×40. a and b, different letters indicate significant differences (P < 0.05).