This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ridoux, O.
Right arrow Articles by Drancourt, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ridoux, O.
Right arrow Articles by Drancourt, M.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, December 1998, p. 3301-3303, Vol. 42, No. 12
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

In Vitro Susceptibilities of the Microsporidia Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis to Albendazole and Its Sulfoxide and Sulfone Metabolites

Olivier Ridoux and Michel Drancourt*

Unité des Rickettsies CNRS UPRES-A 6020, Faculté de Médecine, Université de la Méditerranée, 13385 Marseille cedex 05, France

Received 24 July 1998/Returned for modification 8 September 1998/Accepted 28 September 1998

    ABSTRACT
Top
Abstract
Text
References

In vitro comparisons demonstrated that the efficacy of albendazole, albendazole-sulfoxide, and albendazole-sulfone against pathogenic Encephalitozoon species was proportional to the degree of oxidation at a concentration of >10-3 µg/ml. Furthermore, at a concentration of <10-2 µg/ml, benzimidazoles were more effective against Encephalitozoon cuniculi and Encephalitozoon hellem than against Encephalitozoon intestinalis.

    TEXT
Top
Abstract
Text
References

The microsporidia Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis are emerging obligate intracellular pathogens causing infections in human immunodeficiency virus-infected patients (24). Albendazole has been successfully used in encephalitozoonoses (2, 5, 10, 11, 19, 20). However, incomplete response (25) and relapses have also been documented (15, 23, 26). These clinical investigations have been accompanied by a handful of noncomparative laboratory studies; however, all but one of these (14) have not assessed the role of albendazole-sulfoxide and albendazole-sulfone active metabolites (8). We therefore compared the experimental activities of albendazole, albendazole-sulfoxide, and albendazole-sulfone on Encephalitozoon species in MRC5 cell culture.

The three benzimidazoles (SmithKline Beecham, Nanterre, France) were dissolved at 1 mg/ml in sterile dimethyl sulfoxide (DMSO), and 10-fold serial dilutions (100 to 10-4 µg/ml) were prepared in minimum essential medium (MEM) (Eurobio, Paris, France). Spores of E. cuniculi, E. hellem, and E. intestinalis strains (kindly provided by T. van Gool, University of Amsterdam, Amsterdam, The Netherlands) were cocultivated with MRC5 embryonic lung fibroblasts (BioMérieux, Lyon, France) in a mixture of MEM, 1% glutamine, and 10% heat-inactivated fetal calf serum (Flow Laboratories, Paris, France) at 35°C in a 5% CO2 atmosphere. The infection rate was monitored by rapid-heating Gram-chromotrope staining (21) and microscopic counting of spores in a Kova-slide (Hycor Biomedical, Inc., Irvine, Calif.). For antimicrosporidian activity assays, MRC5 cells subcultured at confluence on glass coverslips in a 24-well microplate were incubated with 100 µl of a suspension of 106 spores/ml for 5 h at 37°C. One milliliter of culture medium, with or without benzimidazole, was then added, and the plate was incubated at 37°C in a 5% CO2 incubator for 6 days. For each plate, a benzimidazole-free positive control and benzimidazole dilutions of 1 to 10-4 µg/ml were tested. After incubation, the mean and standard deviation of the number of microsporidia per field were determined by microscopic examination of rapidly heated Gram-chromotrope-stained coverslips (20 fields observed). The percentage of microsporidian growth inhibition was calculated as [1 - (mean number of infected cells in replicate cultures with benzimidazole/mean number of infected cells in control cultures)] × 100 (± standard error). The benzimidazole concentration inhibiting 90% of microsporidian growth in a control culture (IC90) was estimated from plots of spore number versus log benzimidazole concentration. Each benzimidazole was tested in triplicate. The potential toxicities of benzimidazole and DMSO on MRC5-cultured cells were examined by using a microplaque colorimetric assay adaptated from that previously reported (22). Analysis of variance was used to compare the median and standard deviation values of optical density in the toxicity test and to compare the percentages of growth inhibition for each microsporidian species and for each concentration of each benzimidazole. The Kruskall-Wallis test was used when variances were not homogeneous according to Bartlett's test.

DMSO at a final concentration of 10-3 µg/ml had no toxic effect on MRC5 cells, whereas a statistically significant, moderate toxic effect was observed with albendazole at 100 to 10-4 µg/ml, with albendazole-sulfoxide at 100 µg/ml, and with albendazole-sulfone at 100 to 10-1 µg/ml (P < 0.05) (Table 1). Any benzimidazole concentration tested was significantly effective in inhibiting the growth of any of the three Encephalitozoon species (P < 0.0001) (Table 1). The percentage of E. hellem growth inhibition varied from 97.34 to 90.68% for albendazole, from 96.39 to 92.9% for albendazole-sulfoxide, and from 98.78 to 91.73% for albendazole-sulfone. The IC90s were 2.7 × 10-4 µg/ml for albendazole and <10-4 µg/ml for albendazole-sulfoxide and -sulfone. The percentages of E. cuniculi growth inhibition varied from 97.29 to 53% for albendazole, from 96 to 75.98% for albendazole-sulfoxide, and from 100 to 71.63% for albendazole-sulfone. The IC90s were 3.3 × 10-2 µg/ml for albendazole, 6 × 10-3 µg/ml for sulfoxide, and 2 × 10-3 µg/ml for sulfone. The percentages of E. intestinalis growth inhibition varied from 100 to 29.16% for albendazole, from 100 to 53.43% for albendazole-sulfoxide, and from 98.94 to 50.39% for albendazole-sulfone. The IC90s were 7.1 × 10-2 µg/ml for albendazole, 3.8 × 10-2 µg/ml for sulfoxide, and 10-1 µg/ml for sulfone.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 1.   Intracellular efficacy of albendazole and its sulfoxide and sulfone metabolites on microsporidial growth and estimation of MRC5 host cell viability

Statistical analysis indicated that albendazole-sulfoxide and albendazole-sulfone at concentrations >10-4 µg/ml were significantly (5 to 15 times) more effective than albendazole against E. intestinalis, and albendazole-sulfone was significantly (5 times) more effective than albendazole-sulfoxide (P < 0.05) against any of the three species. E. intestinalis was significantly less susceptible than the two other microsporidian species, regardless of the benzimidazole concentration (P < 0.0001). For benzimidazole concentrations <10-2 µg/ml, E. cuniculi was significantly less susceptible than E. hellem (P < 0.0001).

The previously unreported toxicity for albendazole and, to a lesser extent, its metabolites, we observed could be due to the fact that the microplaque colorimetric assay we used is highly sensitive as a result of the large number of measurements collected for individual benzimidazole concentrations. This moderate toxic effect had a minimal effect on the IC90 determinations. Previous IC determinations of albendazole activity against Encephalitozoon spp. were 2.5 to 0.008 µg/ml for E. cuniculi (1, 4, 9, 16, 27), 0.005 to 0.008 µg/ml for E. intestinalis (3, 14), and 0.008 µg/ml for E. hellem (4). The higher ICs we observed may have resulted from the experimental model we used, which included simultaneous inoculation and treatment; previously, albendazole had been added to well-established cultures. We found E. intestinalis to be less susceptible to albendazole and its two major derivatives than E. hellem and E. cuniculi. The new data were obtained as the three Encephalitozoon species were tested in parallel. Previous studies have included only one species, thus preventing the accurate comparison of interspecies susceptibilities. Albendazole acts by disrupting microsporidian microtubules through beta -tubulin binding (17). Even if six beta -tubulin residues identified as being predictive for benzimidazole susceptibility in parasites (13) exhibited a sequence predictive of susceptibility in E. cuniculi, E. hellem (7), and E. intestinalis (6), divergences in primary sequences may support differences in susceptibility. Alternative hypotheses include E. intestinalis-related altered intracellular penetration or metabolism of albendazole. We also confirmed that albendazole-sulfone is significantly more effective against the Encephalitozoon species than albendazole-sulfoxide, which in turn is more effective than albendazole. E. intestinalis has previously been found to be 1.7 times more susceptible to albendazole-sulfoxide than to albendazole (14), but no data have been presented for albendazole-sulfone. Indeed, in patients treated with oral albendazole, albendazole and albendazole-sulfone remained undetectable (i.e., <0.02 µg/ml) (18), whereas albendazole-sulfoxide concentrations varied between 0.1 and 0.5 µg/ml (12, 18).

    ACKNOWLEDGMENTS

We acknowledge Hervé Tissot Dupont for assistance with statistical tests and Richard Birtles for review of the manuscript.

    FOOTNOTES

* Corresponding author. Mailing address: Unité des Rickettsies CNRS UPRES-A 6020, Faculté de Médecine, 27 Boulevard Jean Moulin, 13385 Marseille cedex 05, France. Phone: 33(0)4 91 32 43 75. Fax: 33 (0)4 91 83 03 90. E-mail: Michel.Drancourt{at}medecine.univ-mrs.fr.

    REFERENCES
Top
Abstract
Text
References

1. Beauvais, B., C. Sarfati, S. Challier, and F. Derouin. 1994. In vitro model to assess effect of antimicrobial agents on Encephalitozoon cuniculi. Antimicrob. Agents Chemother. 38:2440-2448[Abstract/Free Full Text].
2. De Groote, M. A., G. Visvesvara, M. L. Wilson, N. J. Pieniazek, S. B. Slemenda, A. J. daSilva, G. J. Leitch, R. T. Bryan, and R. Reves. 1995. Polymerase chain reaction and culture confirmation of disseminated Encephalitozoon cuniculi in a patient with AIDS: successful therapy with albendazole. J. Infect. Dis. 171:1375-1378[Medline].
3. Didier, E. S. 1997. Effects of albendazole, fumagillin, and TNP-470 on microsporidial replication in vitro. Antimicrob. Agents Chemother. 41:1541-1546[Abstract].
4. Ditrich, O., Z. Kucerova, and B. Koudela. 1994. In vitro sensitivity of Encephalitozoon cuniculi and E. hellem to albendazole. J. Eukaryot. Microbiol. 41:37S[Medline].
5. Dore, G. J., D. J. Marriott, M. C. Hing, J. L. Harkness, and A. S. Field. 1995. Disseminated microsporidiosis due to Septata intestinalis in nine patients infected with the human immunodeficiency virus: response to therapy with albendazole. Clin. Infect. Dis. 21:70-76[Medline].
6. Edling, T., S. Katiyar, G. Visvesvara, and J. Li. 1996. Evolutionary origins of microsporidia and basis for benzimidazole sensitivity: an update. J. Eukaryot. Microbiol. 43:109S[Medline].
7. Edling, T., G. Visvesvara, J. Li, and S. Katiyar. 1994. Cryptosporidium and microsporidial b-tubulin sequences: predictions of benzimidazole sensitivity and phylogeny. J. Eukaryot. Microbiol. 41:38S[Medline].
8. Edwards, G., and A. M. Breckenridge. 1988. Clinical pharmacokinetics of antihelminthic drugs. Clin. Pharmacokinet. 15:67-93[Medline].
9. Franssen, F. F. J., J. T. Lumeij, and F. van Knapen. 1995. Susceptibility of Encephalitozoon cuniculi to several drugs in vitro. Antimicrob. Agents Chemother. 39:1265-1268[Abstract].
10. Franzen, C., A. Müller, P. Hartmann, M. Kochanek, V. Diehl, and G. Fätkenheuer. 1996. Disseminated Encephalitozoon (Septata) intestinalis infection in a patient with AIDS. N. Engl. J. Med. 335:1610-1611[Free Full Text].
11. Gunnarson, G., D. Hurlbut, P. C. DeGirolami, M. Federman, and C. Wanke. 1995. Multiorgan microsporidiosis: report of five cases and review. Clin. Infect. Dis. 21:37-44[Medline].
12. Hoaksey, P. E., K. Awadzi, S. A. Ward, P. A. Coventry, M. E. Orme, and G. Edwards. 1991. Rapid and sensitive method for the determination of albendazole and albendazole sulfoxide in biological fluids. J. Chromatogr. 566:244-249[Medline].
13. Katiyar, S. K., and T. D. Edling. 1994. beta -Tubulin genes of Trichomonas vaginalis. Mol. Biochem. Parasitol. 64:33-42[Medline].
14. Katiyar, S. K., and T. D. Edlind. 1997. In vitro susceptibilities of the AIDS-associated microsporidian Encephalitozoon intestinalis to albendazole, its sulfoxide metabolite, and 12 additional benzimidazole derivatives. Antimicrob. Agents Chemother. 41:2729-2732[Abstract].
15. Lecuit, M., E. Oksenhendler, and C. Sarfati. 1994. Use of albendazole for disseminated microsporidian infection in a patient with AIDS. Clin. Infect. Dis. 19:332-333[Medline].
16. Leitch, G. J., M. Scanlon, A. Shaw, G. S. Visvesvara, and S. Wallace. 1997. Use of a fluorescent probe to assess the activities of candidate agents against intracellular forms of Encephalitozoon microsporidia. Antimicrob. Agents Chemother. 41:337-344[Abstract].
17. Li, J., S. K. Katiyar, A. Hamelin, G. S. Visvesvara, and T. D. Edling. 1996. Tubulin genes from AIDS-associated microsporidia and implications for phylogeny and benzimidazole sensitivity. Mol. Biochem. Parasitol. 78:289-295[Medline].
18. Marriner, S. E., D. L. Morris, B. Dickson, and J. A. Bogan. 1986. Pharmacokinetics of albendazole in man. Eur. J. Clin. Pharmacol. 30:705-708[Medline].
19. Molina, J.-M., C. Chastang, J. Goguel, J.-F. Michiels, C. Sarfati, I. Desportes-Livage, J. Horton, F. Derouin, and J. Modaï. 1998. Albendazole for treatment and prophylaxis of microsporidiosis due to Encephalitozoon intestinalis in patients with AIDS: a randomized double-blind controlled trial. J. Infect. Dis. 177:1373-1377[Medline].
20. Molina, J.-M., E. Oksenhendler, B. Beauvais, C. Sarfati, A. Jaccard, F. Derouin, and J. Modaï. 1995. Disseminated microsporidiosis due to Septata intestinalis in patients with AIDS: clinical features and response to albendazole therapy. J. Infect. Dis. 171:245-249[Medline].
21. Moura, H., D. A. Schwartz, F. Bornay-Llinares, F. C. Sodré, S. Wallace, and G. S. Visvesvara. 1997. A new and improved quick-hot Gram chromotrope technique that differentially stains microsporidian spores in clinical samples, including paraffin-embeded tissues sections. Arch. Pathol. Lab. Med. 121:888-893[Medline].
22. Raoult, D., P. Rousselier, G. Vestris, and J. Tamalet. 1987. In vitro susceptibility of Rickettsia rickettsii and Rickettsia conorii: plaque assay and microplaque colorimetric assay. J. Infect. Dis. 155:1059-1062[Medline].
23. Rossi, R. M., C. Wanke, and M. Federman. 1996. Microsporidian sinusitis in patients with the acquired immunodeficiency syndrome. Laryngoscope 106:966-971[Medline].
24. Weber, R., R. T. Bryan, D. A. Schwartz, and R. L. Owen. 1994. Human microsporidial infections. Clin. Microbiol. Rev. 7:426-461[Abstract/Free Full Text].
25. Weber, R., P. Deplazes, M. Flepp, A. Mathis, R. Baumann, B. Sauer, H. Kuster, and R. Luthy. 1997. Cerebral microsporidiosis due to Encephalitozoon cuniculi in a patient with human immunodeficiency virus infection. N. Engl. J. Med. 336:474-478[Free Full Text].
26. Weber, R., B. Sauer, M. A. Spycher, P. Deplazes, R. Keller, R. Ammann, J. Briner, and R. Lüthy. 1994. Detection of Septata intestinalis in stool specimens and coprodiagnostic monitoring of successful treatment with albendazole. Clin. Infect. Dis. 19:342-345[Medline].
27. Weiss, L. M., E. Michalakakis, C. M. Coyle, H. B. Tanowitz, and M. Wittner. 1994. The in vitro activity of albendazole against Encephalitozoon cuniculi. J. Eukaryot. Microbiol. 41:65S[Medline].

Antimicrobial Agents and Chemotherapy, December 1998, p. 3301-3303, Vol. 42, No. 12
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Arslan, A., Arikoglu, H., Paksoy, Y., Odev, K., Koc, O. (2006). Reply.. Am. J. Roentgenol. 186: 1199-1200 [Full Text]  
  • Wasson, K., Peper, R. L. (2000). Mammalian Microsporidiosis. Vet Pathol 37: 113-128 [Abstract] [Full Text]  
  • Ridoux, O., Drancourt, M. (1999). Lack of In Vitro Antimicrosporidian Activity of Thalidomide. Antimicrob. Agents Chemother. 43: 2305-2306 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ridoux, O.
Right arrow Articles by Drancourt, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ridoux, O.
Right arrow Articles by Drancourt, M.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»