Efficacy of nitazoxanide against Cryptosporidium parvum in cell culture and in animal models

Abstract

Nitazoxanide (NTZ), a drug currently being tested in human clinical trials for efficacy against chronic cryptosporidiosis, was assessed in cell culture and in two animal models. The inhibitory activity of NTZ was compared with that of paromomycin (PRM), a drug that is partially effective against Cryptosporidium parvum. A concentration of 10 microg of NTZ/ml (32 microM) consistently reduced parasite growth in cell culture by more than 90% with little evidence of drug-associated cytotoxicity, in contrast to an 80% reduction produced by PRM at 2,000 microg/ml (3.2 mM). In contrast to its efficacy in vitro, NTZ at either 100 or 200 mg/kg of body weight/day for 10 days was ineffective at reducing the parasite burden in C. parvum-infected, anti-gamma-interferon-conditioned SCID mice. Combined treatment with NTZ and PRM was no more effective than treatment with PRM alone. Finally, NTZ was partially effective at reducing the parasite burden in a gnotobiotic piglet diarrhea model when given orally for 11 days at 250 mg/kg/day but not at 125 mg/kg/day. However, the higher dose of NTZ induced a drug-related diarrhea in piglets that might have influenced its therapeutic efficacy. As we have previously reported, PRM was effective at markedly reducing the parasite burden in piglets at a dosage of 500 mg/kg/day. Our results indicate that of all of the models tested, the piglet diarrhea model most closely mimics the partial response to NTZ treatment reported to occur in patients with chronic cryptosporidiosis.

FIG. 1

Log oocyst shedding from C. parvum-infected, anti-IFN-γ-conditioned SCID mice. Three-week-old male SCID mice received a single injection of 1 mg of XMG1.2 2 h prior to oral inoculation with 10⁷ GCH1 oocysts. The level of oocyst shedding in the feces of each mouse was assessed three times per week. Treatment began after day 6 and ended on day 15. Results are presented as the log of the mean number of oocysts shed per group ± the 95% confidence interval (CI) (bars). Each group had seven mice. Symbols: , 100 mg of NTZ/kg/day and 2,500 mg of PRM/kg/day; ▾, 200 mg of NTZ/kg/day and 2,500 mg of PRM/kg/day; ▴, 2,500 mg of PRM/kg/day; , 100 mg of NTZ/kg/day; □, 200 mg of NTZ/kg/day; and ◊, placebo.

FIG. 2

Mucosal infection scores for treatment groups of C. parvum-infected, anti-IFN-γ-conditioned SCID mice. At necropsy, sections were taken from the pyloric region of the stomach, mid-small intestine, ileum, cecum, and proximal colon for histological analysis to determine the extent of mucosal infection. Each site was assigned a score depending on the extent of infection ranging from 0 (no infection) to 5 (extensively infected mucosa). Results are presented as the mean total score of the five sites for each group ± 95% confidence intervals (CI) (bars). When they were tested by Student-Newman-Keuls analysis of variance (α = 0.05), the mucosal scores fell into three sets, with significant differences among them (6 df; F = 18.167; P < 0.001). Mice treated with placebo or with NTZ alone (at 100 or 200 mg/kg/day) formed one distinguishable statistical set (set one), mice treated with 2,500 mg of PRM/kg/day with or without NTZ (100 or 200 mg/kg/day) formed a second distinguishable set (set two), and the uninfected controls formed a third statistical set (set three). The mucosal infection scores for the mice treated with PRM (n = 41) were significantly lower than those for the mice not treated with PRM (n = 42) (7.78 ± 0.38 versus 13.69 ± 0.73, respectively; P < 0.001). In contrast, the analysis of the effects on the mucosal infection score of the presence and absence of NTZ treatment did not show any differences between groups (data not shown).

FIG. 3

Fecal oocyst excretion scores of infected piglets treated with NTZ at either 125 () or 250 (◊) mg/kg/day, placebo (■), or PRM at 500 mg/kg/day (▴). In multiple regression analysis, the oocyst excretion score was found to be significantly related to treatment group, with the highest scores being observed in the placebo group, followed by the lower-dose NTZ group and then the higher-dose NTZ group, and the lowest scores being observed in the PRM group (F = 42.507; P < 0.001). Further comparison revealed that, during days 7 through 13, the scores for the piglets treated with NTZ at 250 mg/kg/day were significantly lower than those for the placebo-treated piglets (Z = −3.258; P = 0.001, two-tailed Wilcoxon signed rank test). In contrast, subgroup comparison of the infected placebo-treated control piglets and piglets treated with NTZ at 125 mg/kg/day did not reveal any significant difference. Oocyst shedding was significantly less marked in the PRM-treated group than in any of the other groups (P < 0.001). Values are means ± standard errors of the means (SEM).

FIG. 4

Intestinal mucosal scores for 24 piglets euthanized on day 13 of the experiment. The line represents a quadratic least-squares fit of the data (R² = 0.64849; P < 0.001). Analysis of variance revealed that the mucosal score varied significantly from group to group (F = 22.21; P < 0.0001). Comparisons made by using the Mann-Whitney U test and Wilcoxon rank sum test revealed that the scores were significantly different for the following pairs of groups: NTZ at 250 mg/kg/day and PRM (P = 0.050), infected controls and NTZ at 250 mg/kg/day (P = 0.025), infected controls and PRM (P = 0.014), and infected controls and uninfected controls (P = 0.014). The score for the group treated with NTZ at 125 mg/kg/day was not significantly different from that for the infected control group (P = 0.282). 3 and 4 denote that three and four piglets, respectively, had the same scores.