1 Escola Superior de Tecnologia da Saúde do Porto, Rua João Oliveira Ramos 87, 4000-294 Porto; 2 Instituto de Ciências Biomédicas de Abel Salazar, 4099-003 Porto; 3 Laboratório de Microbiologia, Faculdade de Farmácia da Universidade do Porto, 4050-047 Porto, Portugal
Received 11 January 2002; returned 15 May 2002; revised 24 September 2002; accepted 13 January 2003
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Abstract |
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Keywords: Giardia, susceptibility assays, metronidazole, albendazole
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Introduction |
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There is only a limited range of drugs available for treatment of giardiasis. These drugs comprise the nitroimidazoles, quinacrine and furazolidone. Metronidazole is the drug of choice, but alternatives, such as albendazole, are available.57
Some clinical reports suggest the appearance of drug resistance to explain treatment failure in giardiasis.7
The establishment of G. lamblia axenic cultures in Portugal made it possible to evaluate the susceptibility of isolates to antiparasitic drugs. Giardiasis is considered an important parasitic disease in Portugal, but there is a lack of information regarding the antimicrobial susceptibility profile of the strains, which parasitize the Portuguese population. In the present study, we describe the isolation and axenic cutting of G. lamblia parasites from human faeces obtained in the Porto region and report on the susceptibility of these isolates to metronidazole and albendazole using the inhibition of adherence method.
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Materials and methods |
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Fifty-three human faecal samples (13 days old) from different patients, containing abundant G. lamblia cysts, were obtained from an epidemiological survey of the school children population and clinical laboratories in the Porto region.
G. lamblia cysts were purified and concentrated from faeces combining the sucrose flotation method with a simplified sucrose gradient method.4 The cysts, after being washed twice in distilled water, were resuspended in distilled water and stored at 4°C for a maximum of 3 days prior to use.
Excystation and axenization
The excystation procedure was a modification of the Bingham & Meyer technique performed by Schupp et al.2 The isolation procedure involved three steps: the concentration and cleaning of cysts by centrifugation in sucrose gradients performed from 1 to 3 days after collection, the induction of excystation performed in acid solution from 1 to 5 days after cleaning cyst suspensions, and the culture and axenization in modified TYI-S-33 medium.
Cryopreservation of G. lamblia trophozoites was performed using the method of Hautus et al.4
The control reference isolates were G. lamblia ATCC 30888 and ATCC 30957.
Chemotherapeutic agents
The chemotherapeutic agents used were metronidazole and albendazole (Sigma). Stock solutions were prepared in phosphate-buffered saline (10 mM in phosphate, pH 7.4), for metronidazole, and DMSO for albendazole. The final DMSO concentration in the culture tubes was always <0.5% (v/v).
Adherence inhibition assays
To assess trophozoite susceptibility, the adherence inhibition method was performed.6
The results are presented as a percentage of inhibition of adherence (%IA) according to the formula:
%IA = (control cell number tested cell number)/control cell number x 100
All the assays were performed in duplicate and repeated twice for each isolate and drug.
Statistical analysis
Probit analysis was used to calculate the concentration of drug that inhibited the viability by 50% of G. lamblia adherent trophozoites (IC50).
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Results |
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The susceptibility of the 18 G. lamblia isolates to metronidazole and albendazole was evaluated by the inhibition of adherence method.6
The IC50 values calculated for each isolate after 24 h of drug exposure are presented in Table 1. For metronidazole the IC50 values varied from 2.38 to 11.50 µM (mean 4.92 µM; S.D. 2.56 µM), representing a range of variation of 4.8-fold in susceptibility. For albendazole, the range of IC50 values varied from 0.027 to 0.192 µM (mean 0.088 µM; S.D. 0.035 µM), representing a 7.1-fold variation. The isolates were 56 times more susceptible to albendazole than to metronidazole.
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Discussion |
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Our isolation rate of 34% is similar to rates described in the literature, varying from 21% to 44%.4,8 Cyst viability, microbial contamination and adaptation of trophozoites to the medium have been described as the major causes for the low rate of excystation.8 In our case, we increased cyst inoculum and the antimicrobial drug concentration to try to achieve a higher isolation rate, but these changes failed to produce higher values. It is possible that genetic variation is one important reason for the inability of excysted trophozoites to survive in TYI-S-33 medium, as suggested by Meloni & Thompson;8 they failed to establish cultures of excysted trophozoites isolated from dogs.
The principal objective of our study was to obtain susceptibility data for metronidazole and albendazole against G. lamblia isolates from the Portuguese population. For this purpose, we used the adherence properties of G. lamblia trophozoites instead of the capacity of cells to divide. The ability to adhere to gastrointestinal mucosa is an important prerequisite for G. lamblia to establish a sustainable infection.1 In the presence of active antiparasitic drugs in vivo, trophozoites fail to adhere to epithelial layers, thus helping terminate the infection.
Our results indicate that the 18 G. lamblia isolates were susceptible to metronidazole and albendazole. Several authors obtained similar levels of susceptibility in spite of differences in the methodology used, such as the time the parasites were in contact with the drug.3,9 The IC50 and doseresponse curves of our isolates show small variation among the 18 isolates and these values are close to the ATCC reference isolates (Table 1). Similar results were obtained by Farbey et al.3 for albendazole using 29 Australian G. lamblia isolates, but against metronidazole the IC50 values showed greater variation, in contrast to our results. Drug presence itself can be responsible for such variation, since, in vitro, cultivation of G. lamblia isolates in the presence of sublethal concentrations of metronidazole resulted in an increase in resistance to the drug.10
We conclude that metronidazole is effective for the treatment of giardiasis in our population and that albendazole will be a suitable alternative in the future.
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Acknowledgements |
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Footnotes |
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References |
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2 . Schupp, D., Januschka, M., Sherlock, L., Stibbs, H., Meyer, E., Bemrick, W. et al. (1988). Production of viable Giardia cysts in vitro: determination by fluorogenic dye staining, excystation, and animal infectivity in the mouse and Mongolian gerbil. Gastroenterology 95, 110.[ISI][Medline]
3 . Farbey, M. D., Reynoldson, J. A. & Thompson, R. C. A. (1995). In vitro drug susceptibility of 29 isolates of Giardia duodenalis from humans as assessed by an adhesion assay. International Journal for Parasitology 25, 5939.[CrossRef][ISI][Medline]
4 . Hautus, M., Kortbeek, L., Vetter, J. & Laarman, J. (1988). In vitro excystation and subsequent axenic growth of Giardia lamblia. Transactions of The Royal Society of Tropical Medicine and Hygiene 82, 85861.[CrossRef][ISI][Medline]
5 . Romero-Cabello, R., Robert, L., Munoz-Garcia, R. & Tanaka, J. (1995). Randomized study comparing the safety and efficacy of albendazole and metronidazole in the treatment of giardiasis in children. Revista Latinoamericana de Microbiologia 37, 31523.[Medline]
6 . Meloni, B. P., Thompson, R. C. A., Reynoldson, J. & Seville P. (1990). Albendazole, a more effective antigiardial agent in vitro than metronidazole or tinidazole. Transactions of The Royal Society of Tropical Medicine and Hygiene 84, 3759.[ISI][Medline]
7 . Nash, T. E., Ohl, C. A., Thomas, E., Subramanian, G., Keiser, P. & Moore, T. A. (2001). Treatment of patients with refractory giardiasis. Clinical Infectious Diseases 33, 228.[CrossRef][ISI][Medline]
8 . Meloni, B. P. & Thompson, R. C. A. (1987). Comparative studies on the axenic in vitro cultivation of Giardia of human and canine origin: evidence for intraspecific variation. Transactions of The Royal Society of Tropical Medicine and Hygiene 81, 63740.[CrossRef][ISI][Medline]
9 . Cedillo-Rivera, R. & Muñoz, O. (1992). In-vitro susceptibility of Giardia lamblia to albendazole, mebendazole and other chemotherapeutic agents. Journal of Medical Microbiology 37, 2214.[Abstract]
10 . Townson, S. M., Laqua, H., Upcroft, P., Boreham, P. F. & Upcroft, J. A. (1992). Induction of metronidazole and furazolidone resistance in Giardia. Transactions of The Royal Society of Tropical Medicine and Hygiene 86, 5212.[ISI][Medline]
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