Isolation, excystation and axenization of Giardia lamblia isolates: in vitro susceptibility to metronidazole and albendazole

Agostinho Cruz1,2,*, M. Isaura Sousa3, Zaida Azeredo2, Emilia Leite1, J. C. Figueiredo de Sousa3 and Miguel Cabral3

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
From 53 samples of human faeces containing Giardia lamblia cysts, 18 isolates were successfully excysted in vitro, and cultivated axenically in TYI-S-33 modified medium. The in vitro effects of metronidazole and albendazole on these isolates were evaluated by the trophozoite adherence inhibition method. The IC50 was between 2.4 and 11.5 µM for metronidazole and 0.027 and 0.192 µM for albendazole. These IC50 values were similar to those found for the ATCC 30888 and 30957 reference isolates. All isolates were susceptible to the antiparasitic drugs tested. These results suggest that resistance of G. lamblia to metronidazole and albendazole does not seem to be a significant problem in our population.

Keywords: Giardia, susceptibility assays, metronidazole, albendazole


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There is a lack of information about Giardia lamblia strains, which parasitize different populations. The difficulties in isolating and cultivating G. lamblia trophozoites in vitro are probably the main reason. In the 1970s, in Portland, G. lamblia was isolated and cultured axenically for the first time (ref. P-1), and was kept as a reference in the American Type Culture Collection (ATCC 30888).1 Most in vitro studies of G. lamblia have used the P-1 or the WB isolate (ATCC 30957), which was obtained from a patient with prolonged symptomatic giardiasis, probably acquired in Afghanistan.1 The introduction of methods for the axenic cultivation of G. lamblia, improvements in culture media and development of techniques for the in vitro excystation of the parasite have made it possible to obtain many axenic isolates from various geographical regions.2,3 Isolation can also be performed using trophozoites obtained directly from duodenal aspiration or from excystation in vivo.2,4

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
G. lamblia cyst isolation

Fifty-three human faecal samples (1–3 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).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
From 53 different human faecal samples containing G. lamblia cysts, we were able to obtain 18 G. lamblia axenic cultures, representing an overall success rate of 34%. A high concentration of cysts in the faeces was needed for successful excystation and axenization (Figure 1). During the process of excystation, trophozoites hatched from their cysts, underwent cytokinesis and attached to the surface of the culture tubes 1–3 h after in vitro excystation. In successful cultures, trophozoites began to divide and multiply within 2–3 days and subsequently formed a monolayer on the surface of the culture tube.



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Figure 1. Differential interference contrast photomicrographs of excystation and axenic culture of G. lamblia in TYI-S-33 modified medium. (a) Cysts prior to excystation; (b–e) excystation phases; (f) axenic monolayer.

 
A significant number of purified cyst samples were not established as cultures. The three major reasons for this were: no excystation, persistent microbial contamination and failure of the trophozoites to establish the monolayer culture. In the latter case, the number of trophozoites increased during the first 24 h, but then declined.

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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Table 1.  Susceptibility of G. lamblia isolates to the drugs tested
 
The mean IC50 values for metronidazole and albendazole obtained for the 18 isolates in the inhibition of adherence assay are similar to the mean values obtained with the ATCC reference isolates.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This report of the isolation, excystation and axenic culture of G. lamblia parasites is an important step in establishing drug susceptibility data from the Portuguese population. The methodology used simulates the intestinal passage of the cyst.2 It includes low pH induction in a reducing environment followed by neutralization and incubation in medium and is a good alternative to duodenal aspiration or in vivo excystation. Duodenal aspiration is not readily accepted, or justified in most cases, particularly in asymptomatic infections. Excystation in vivo requires colonies of Giardia-free animals, normally gerbils, which in spite of the advantage of large numbers of trophozoites produced, is expensive, time-consuming and not easy to perform.

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 dose–response 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.


    Acknowledgements
 
This work was supported by ‘Comissão de Fomento da Investigação em Cuidados de Saúde, Project 235/97, Ministério da Saúde, Portugal’.


    Footnotes
 
* Corresponding author. Tel: +351-225-081-300; Fax: +351-226-180-627; E-mail: agostinho-cruz{at}clix.pt Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Adam, R. D. (2001). Biology of Giardia lamblia. Clinical Microbiology Reviews 55, 706–32.

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, 1–10.[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, 593–9.[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, 858–61.[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, 315–23.[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, 375–9.[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, 22–8.[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, 637–40.[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, 221–4.[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, 521–2.[ISI][Medline]





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