a Laboratoire d'Immunologie et Immunopathologie, UPRES-EA 2128, CHU, 14033 Caen; b Laboratoire de Parasitologie Expérimentale, UPRES-JE 2008, CHU, 76000 Rouen, France
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Abstract |
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Introduction |
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The aim of this study was to investigate in vitro the efficacy of nitazoxanide, tizoxanide and tizoxanide glucuronide on sporozoite invasion and asexual and sexual development of C. parvum in the enterocytic cell line HCT-8. Asexual and sexual stages of parasite development were assessed by both enzyme immunoassay and immunofluorescence.7,8
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Materials and methods |
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HCT-8 cells (CCL-244) were obtained from the American Type Culture Collection, Rockville, MD, USA. They were maintained in culture in BHK 21 (Eagle's modified Dulbecco's) medium supplemented with 1% non-essential amino acids and 20% fetal calf serum (Gibco BRL, Gaithersburg, MD, USA) in a 5% CO2 humidified atmosphere. HCT-8 cells were cultured in 96-well Nunclon microplates (Nunc, Naperville, IL, USA) at 1.5 x 105 cells/well. Cultures were simultaneously performed in 16-well tissue culture chambers (LabTek Chamber Slides; Nunc).
C. parvum sporozoites were obtained from the faeces of experimentally infected calves (kindly provided by Dr Naciri, Laboratoire de Pathologie Aviaire, INRA, Nouzilly, France) as previously described.9 Briefly, C. parvum oocysts were concentrated on a sucrose gradient and then excysted using a 1.5% taurocholate solution. Culture medium was removed from 24 h HCT-8 cultures and 100 µL per well of BHK 21 medium containing 1.52 x 105 freshly isolated sporozoites was added to cell monolayers for 2 h at 37°C. Two hundred microlitres of BHK 21 medium supplemented with paraminobenzoic acid 4 mg/L (Sigma, St Louis, MO, USA), ascorbic acid 35 mg/L (Sigma), glucose 25 mmol/L (Merck, Darmstadt, Germany), insulin 100 IU/L (Novo Nordisk Pharmaceutique, BoulogneBillancourt, France), HEPES 15 mmol/L (Sigma), streptomycin 500 mg/L (Gibco BRL), penicillin 105 IU/L (Gibco BRL) and fetal calf serum 20% (v/v) was added to each well, and cells were cultured for an additional 46 h.10,11
Enzyme immunoassay (EIA) for the detection of C. parvum in HCT-8 cultures was performed as previously described8 using rat polyclonal antibodies against Cryptosporidium and biotinSP-conjugated anti-rat IgG and IgM (heavy and light chain) goat F(ab')2 fragments 2 mg/L (Jackson Immunoresearch, Westgrove, PE, USA) as secondary antibody revealed using alkaline phosphatase-coupled avidin (ABC Reagent; Vector Laboratories, Burlingame, CA, USA).
Inhibitory activities of agents are expressed as percentages, the per cent inhibition was defined as
(A405 in infected cells with agent)
1 (A405 in uninfected cells with agent) x 100
(A405 in infected cells without agent)
(A405 in uninfected cells without agent)
The MIC50 was defined as the concentration (in mg/L of culture) of agent that resulted in a 50% inhibition of C. parvum development.
The cytotoxicities of nitazoxanide, tizoxanide and tizoxanide glucuronide were determined using trypan blue exclusion and nitroblue tetrazolium chloride monohydrate reduction assays (CellTiter 96 AQueous non-radioactive cell proliferation assay; Promega, Madison, WI, USA). Controls included infected and uninfected cells in culture medium. Results were expressed as the decrease in absorbance at 450 nm (A450) expressed as percentages of the A450 values in control cultures. From preliminary studies we verified that HCT-8 cell cytotoxicity was minimal (decrease of A450 from 0 to 14%) for all controls and at all concentrations of agents, except nitazoxanide at 25 and 50 mg/L (A450 decrease of 36% and 39%, respectively).
A405 values in control cultures in which heat-inactivated sporozoites were added instead of viable sporozoites, were not significantly different from A405 values in the absence of heat-inactivated sporozoites. For uninfected wells, one s.d. accounted for <10% and 15% of the mean A405 for each microplate and for the pooled data of five microplates, respectively. For infected wells, one s.d. accounted for <10% and 21% of the mean A405 for each microplate and for the pooled data of five microplates, respectively.
In the EIA, background A405, resulting from the intense colour of the solutions, was higher in uninfected wells exposed to drugs than in control wells without agent, and for this reason they were used as controls for infected wells containing the same agent concentration. In addition, disruption of infected monolayers and/or peeling off of HCT-8 cells occurred during EIA in wells containing tizoxanide or tizoxanide glucuronide at 1050 mg/L. For tizoxanide and tizoxanide glucuronide, high background A405 was observed at concentrations of >50 mg/L, preventing any conclusions from being drawn.
The effects of nitazoxanide, tizoxanide and tizoxanide glucuronide on the various stages of the C. parvum life cycle were studied by adding the tested compounds, at concentrations ranging from 1 to 50 mg/L, at the start of the culture (sporozoite stage), 2 h after adding sporozoites (trophozoite stage) and 18 h after adding sporozoites (sexual stages).
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Results |
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Discussion |
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Previous clinical studies have shown that nitazoxanide is clinically active against cryptosporidiosis.13,14 This compound has a wide range of antimicrobial activities, particularly against intestinal protozoa and helminths.15 Present data underline the potential interest in nitazoxanide metabolites as candidates for in vivo studies. Interestingly, studies in an immunosuppressed rat model suggest that relapses are less frequent after treatment with nitazoxanide than with the non-absorbable paromomycin and sinefungin (Li, X., unpublished results). Taken together, these data indicate the need for additional studies on the biliary clearance of nitazoxanide metabolites and their potential activity on biliary tract cryptosporidiosis.
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Notes |
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References |
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2
.
Kelly, P., Davies, S. E., Mandanda, B., Veitch, A., McPhail, G., Zulu, I. et al. (1997). Enteropathy in Zambians with HIV related diarrhoea: regression modelling of potential determinants of mucosal damage. Gut 41, 8116.
3 . Blagburn, B. B. L. & Soave, R. (1997). Prophylaxis and chemotherapy: human and animal. In Cryptosporidium and Cryptosporidiosis, (Fayer, R., Ed.), pp. 11127. CRC Press, Boca Raton, FL.
4 . Tzipori, S. (1998). Cryptosporidiosis: laboratory investigations and chemotherapy. Advances in Parasitology 40, 187221.[ISI][Medline]
5 . Bissuel, F., Cotte, L., Rabodonirina, M., Rougier, P., Piens, M. A. & Trepo, C. (1994). Paromomycin: an effective treatment for cryptosporidial diarrhea in patients with AIDS. Clinical Infectious Diseases 18, 4479.[ISI][Medline]
6 . Stockis, A., Lins, R., Deroubaix, X., Jeanbaptiste, B., Calderon, P. & Rossignol, J. F. (1996). Pharmacokinetics of nitazoxanide after single oral dose administration in 6 healthy volunteers. International Journal of Clinical Pharmacology and Therapeutics 34, 34951.[ISI][Medline]
7 . Egraz-Bernard, M., Favennec, L., Agnamey, P., Ballet, J. J. & Brasseur, P. (1996). Inhibition of complete development of Cryptosporidium parvum in Caco-2 cells. European Journal of Clinical Microbiology and Infectious Diseases 15, 897900.[ISI][Medline]
8 . Gargala, G., Delaunay, A., Favennec, L., Brasseur, P. & Ballet, J. J. (1999). Enzyme immunoassay detection of Cryptosporidium parvum inhibition by sinefungin in sporozoite infected HCT-8 enterocytic cells. International Journal for Parasitology 29, 7039.[ISI][Medline]
9 . Buraud, M., Forget, E., Favennec, L., Bizet, J., Gobert, J. G. & Deluol, A. M. (1991). Sexual stage development of cryptosporidia in the Caco-2 cell line. Infection and Immunity 59, 46103.[ISI][Medline]
10 . Maillot, C., Favennec, L., François, A., Ducrotte, P. & Brasseur, P. (1997). Sexual and asexual development of Cryptosporidium parvum in five oocyst- or sporozoite-infected human enterocytic cell lines. Journal of Eukaryotic Microbiology 44, 5825.[ISI][Medline]
11 . Upton, S. J., Tilley, M. & Brillhart, D. B. (1995). Effects of select medium supplements on in vitro development of Cryptosporidium parvum in HCT-8 cells. Journal of Clinical Microbiology 33, 3715.[Abstract]
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.
Theodos, C. M., Griffiths, J. K., D'Onfro, J., Fairfield A. & Tzipori, S. (1998). Efficacy of nitazoxanide against Cryptosporidium parvum in cell culture and in animal models. Antimicrobial Agents and Chemotherapy 42, 195965.
13 . Doumbo, O., Rossignol, J. F., Pichard, E., Traore, H. A., Dembele, T. M., Diakite, M. et al. (1997). Nitazoxanide in the treatment of cryptosporidial diarrhea and other intestinal parasitic infections associated with acquired immunodeficiency syndrome in tropical Africa. American Journal of Tropical Medicine and Hygiene 56, 6379.[ISI][Medline]
14 . Rossignol, J. F., Hidalgo, H., Feregrino, M., Higuera, F., Gomez, W. H., Romero, J. L. et al. (1998). A double-blind placebo-controlled study of nitazoxanide in the treatment of cryptosporidial diarrhoea in AIDS patients in Mexico. Transactions of the Royal Society of Tropical Medicine and Hygiene 92, 6636.[ISI][Medline]
15 . Abaza, H., El-Zayadi, A., Kabil, S. M. & Rizk, H. (1998). Nitazoxanide in the treatment of patients with intestinal protozoan and helminthic infections: a report on 546 patients in Egypt. Current Therapeutic Research 59, 11621.[ISI]
Received 19 July 1999; returned 30 November 1999; revised 16 December 1999; accepted 6 February 2000