الفهرس 
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Abstract
Cryptosporidiosis is a disease caused by a coccidian parasite of the genus Cryptosporidium. Cryptosporidium hominis and Cryptosporidium parvum are the most frequent causes of human infection. Humans acquire infection through the feco-oral route, or by drinking contaminated water. Worldwide, cryptosporidiosis infects immunocompetent individuals mostly children under five years of age and immunosuppressed persons particularly those with HIV-infection.
Several drugs have been tested in clinical trials; nitazoxanide, rifabutin, paromomycin, and several macrolides as clarithromycin, azithromycin, and spiramycin with limited effectiveness and variable side effects. Cryptosporidiosis wide distribution triggered the search for new strategies to explore newer agents and overcome the limitations of the current treatment regimens, as effective treatments to eliminate Cryptosporidium are still lacking.
In a review of the recent and probable therapeutic agents available for the treatment of cryptosporidiosis especially among immunosuppressed individuals, nanoparticles were used as anti-parasite agents, improving the distribution, bioavailability and decreasing the toxicity of the present used drugs.
Chitosan is a non-toxic polysaccharide derivative of crabs and shrimp with natural chitin alkaline deacetylation. The usage of chitosan has been stretched to numerous fields because of its numerous advantages (cosmetic industries, agriculture, water treatment, food packing and pharmaceuticals). Chitosan inherent features (chelating capacity, physical state, positive charge density, etc.) are mainly accountable for its antimicrobial actions. It is relatively cheap and non-toxic that guarantees its appropriate application in developing countries. Silver and gold NPs are much more costly for nanotechnology than using the CS NPs.
In the present study, the efficacy of nitazoxanide (NTZ), chitosan nanoparticles (CS NPs) and NTZ loaded CS NPs was evaluated in addition to the aim of developing a safe means and effective drug delivery system against Cryptosporidium infection in both immunosuppressed and immunocompetent mice. CS nanoparticles were prepared by ionotropic gelation method.
The drug efficacy was evaluated using 100 male Swiss Albino mice aged 6 -8 weeks, weighing 20–25 g purchased from Schistosome Biological Supply Center (SBSC), Theodor Bilharz Research Institute (TBRI). Mice were categorised into two main groups; control and treated groups with further subgrouping (each subgroup comprised 10 mice). The control group was subdivided into negative control subgroups A1and A2; noninfected nontreated immunocompetent and immunocompromised respectively and positive control subgroups B1 and B2 immunocompetent and immunocompromised infected nontreated subgroups. Treated subgroups included C1 and C2 immunocompetent and immunocompromised NTZ treated at a dose of 200 mg/kg/day. D1 and D2 immunocompetent and immunocompromised CS treated subgroups at a dose of 20 µg of CS NPs in 200 µl of PBS/mouse/day. Finally, E1 and E2 immunocompetent and immunocompromised NTZ loaded CS NPs (200 mg/kg/day) treated subgroups; for 3 days for immunocompetent subgroups and for 6 days for immunocompromised subgroups. Infection was established using an esophageal tube and 10,000 C. parvum oocysts/mouse were inoculated. Immunosuppression was achieved by giving dexamethasone drug orally at a dose of 0.25 mg/kg/day for 14 days three times per week before C. parvum oocysts inoculation.
One week after inoculation, fresh fecal pellets were collected from each subgroup to check for the presence of oocysts in stools of all infected subgroups. Sacrifice of all surviving mice from all subgroups was done on the 21st day PI.
By clinical observation, infected treated mice continued quite asymptomatic with normal conduct and food intake. Immunocompetent nontreated mice experienced decrease in food intake with reduced activity in addition to marked hair loss, subcutaneous edema, skin lesions and ulcerations in the immunocompromised infected nontreated subgroup. In the meantime, the most substantial improvement in mice behavior and food intake was detected amongst those treated with NTZ loaded CS NPs.
In drug efficacy evaluation results of the current work, mice mortality rates (MR%) and parasitological drug assessment was conducted based on oocysts percent reduction (%R) in both stool and intestinal contents. All used drugs, NTZ, CS NPs and NTZ loaded CS NPs induced a decrease in the MR of infected mice. The lowest MR was that for the infected immunocompetent and immunocompromised subgroups treated with NTZ loaded CS NPs with values of 10% and 30% respectively.
Parasite count and percent reduction (%R) as parasitological means of drug evaluation was conducted 21 days PI. Microscopic stool examination showed that C. parvum oocysts percent reduction in NTZ loaded CS NPs subgroups was 73.51% and 67.14% in the infected immunocompetent and immunocompromised subgroups respectively with high statistical significance (p<0.001).
As for oocyst counts in the examined gut contents, again the percent reduction in the number of C. parvum in the NTZ loaded CS NPs subgroups was the highest (52.80% and 40.40% in infected immunocompetent and immunocompromised mice subgroups respectively). Both values were statistically significant (p<0.001). Blank CS and NTZ showed inferior results.
Cryptosporidium specific serum IgM and IgG antibody response was studied to assess and compare the response to the used drugs in different study subgroups. IgM and IgG antibodies levels increased significantly in infected nontreated immunocompetent and immunocompromised mice subgroups, meanwhile, in immunocompetent and immunocompromised mice receiving NTZ loaded CS NPs, IgM and IgG concentrations decreased significantly (p<0.001) when compared to infected nontreated subgroups.
Conclusively, the above results pointed to the fact that NTZ loaded CS NPs (CS being of non-toxic nature in addition to its modest anti Cryptosporidium effect) gave the highest significant efficacy in all subgroups. This might be promising in the continuing efforts to treat cryptosporidiosis in both immunocompetent and immunocompromised humans.