Highly Active AntiRetroviral Therapy and cryptosporidiosis

In HIV infected persons, Cryptosporidium parvum causes chronic diarrhoea, which can be life-threatening in persons with AIDS and with a low CD4+ T cell count. However, a specific and effective therapy for this opportunistic infection does not yet exist. Since the use of a combination therapy with a highly active antiretroviral therapy (HAART), the prevalence of C. parvum infection in persons with AIDS has been strongly reduced. This favorable outcome was usually attributed to the recovery of the host immunity, however improvements from this opportunistic infection have been demonstrated even in the absence of immunological recovery. The aim of the present study was to determine whether HIV protease inhibitors (PIs) exert an anti-C. parvum activity. We selected the indinavir (an aspartyl protease inhibitor included in HAART) for our experiments, since a resolution of cryptosporidial enteritis in a person with AIDS after treatment with this drug has been reported. Human ileocecal adenocarcinoma tumor cells (HCT-8) were used as in vitro model. To determine whether or not indinavir had an effect on the parasite attachment to, or invasion of the HCT-8 cells, indinavir was added to the cultures at the same time as the infective dose (3 oocysts/cell) at one of the following concentrations: 0.1, 0.5, 5, 10, 20, and 50 microM (maximum DMSO content 0.5% vol/vol). To determine whether or not indinavir had an effect on established C. parvum infection, HCT-8 cells were infected with excysted oocysts at a ratio of 3 oocysts/cell at day 0, and then indinavir at a concentration of 50 microM was added to the cultures every 24 h for 4 days. The infection level was evaluated at 2, 3, 4 and 5 days p.i. using a flowcytometric assay. Three-day-old Balb/c mice were used as animal model, animals were infected per os with 50 microl of PBS containing 10(5) oocysts. The infected mice were divided into two groups (Group A and Group B), both of which received per os indinavir diluted in PBS with 0.1% DMSO at a concentration of 10 microM (24 mg/kg). For Group A, which consisted of 15 mice (3 litters), indinavir was administered at the same time that experimental infection was performed and then every day until the mice were sacrificed (i.e., 5 days p.i.), to determine the effect of indinavir on the attachment/invasion of the enterocytes. For Group B, which also consisted of 15 mice (3 litters), indinavir was administered after the infection was established (i.e., 72 h p.i.) and every day until being sacrificed, to determine the effect of indinavir on established infection. The mice of Group B were sacrificed 7, 10, 11 and 13 days p.i., corresponding to 4, 7, 8, and 10 days of treatment with indinavir. In vitro, the treatment of the excystated oocysts with different concentrations of indinavir reduced the percentage of HCT-8 infected cells in a dose-dependent manner. For established infection, the treatment with 50 microM of indinavir decreased the percentage of infected cells in a time-dependent manner. Treatment for 48 h resulted in a 40.1% reduction in infected cells (from 90% to 53%). After 72 h of treatment, the percentage of infected cells did not substantially differ from that observed after 48 h. Treatment for 96 h resulted in a 57.8% reduction (from 90 to 38%). In vivo, mice treated with indinavir at the same time they were infected with the oocysts showed a 93% reduction in the number of oocysts present in the entire intestinal contents and a 91% reduction in the number of intracellular parasites in the ileum. For established infection, indinavir treatment reduced the number of oocysts in the entire intestinal content by about 50% and the number of intracellular parasites in the ileum by about 70%. These data demonstrate that PIs directly exert an inhibitory effect on C. parvum and the extent of this effect depended on the specific dose and the duration of treatment. Although there are no reports of aspartyl proteases in C. parvum, the inhibitory effect of PIs on C. parvum growth in vitro suggests that aspartyl proteases could have some important functions for this parasite. In fact, proteolytic activities have been demonstrated during peak periods of excystation in C. parvum oocysts and cysteine and serine protease classes have been functionally associated with this process. Moreover, we identified several different C. parvum sequences that showed homology with a protein family related to aspartyl proteases. In prospect, PIs could be valuable for the chemotherapy of cryptosporidiosis.