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Iranian J Parasitol: Vol. 3, No. 2, 2008, pp. 38-42 Original Article

In Vitro Effects of Metronidazole and Albendazole on Giardia
lamblia Isolated from Iranian Patients
F Mohamadnezhad, *F Ghaffarifar, A Dalimi

Dept. of Parasitology, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran

(Received 28 Dec 2007; Accepted 08 Mar 2008)

Abstract
Background: The aim of the present study was to evaluate the effects of metronidazole and albendazole against clinical
isolates of Giardia lamblia in vitro.
Methods: From all human samples of containing cysts, 10 isolates were successfully excysted in vitro. Trophozites viability
was assessed by eosine 0.1% and cultured axenically in TYI-S-33 modified medium supplemented with heat inactivated
bovine serum 10%. All cultures were incubated in 37°C for 24-48 h. After this time trophozoites were exposed to different
concentration (0.05, 0.1, 2, 10, 50 µg/ml) of drugs at 37º for 4 h. The IC50 estimated between 0.1 and 10µg/ml for metroni-
dazole and 0.062 and 0.1 µg/ml for albendazole.
Results: Eight isolates were found susceptible to the metronidazole while all isolates were found susceptible to the albenda-
zole. Statistical results indicated that there was significant difference (P<0.05) in the sensitivity to metronidazole and alben-
dazole in all isolates.
Conclusion: The killing affects of albendazole on G.lamblia was greater than metronidazole.

Keywords: Giardia lamblia, Metronidazole, Albendazole, Susceptibility, In-Vitro, Iran.
Introduction
In human giardiasis, therapeutic failure is oc- curring more and more frequently, due to low iardia lamblia, is a protozoan parasite in compliance with drug therapy, reinfestation or testine that causes extensive mor- parasite resistance to metronidazole and/or the bidity in the worldwide. Giardiasis is an impor- nitroimidazole-related compounds secnidazole, tant cause of chronic diarrhea and malab- tinidazole plus ornidazole as well as quinacrine sorbtion. Giardia infects approximately 2% of and furazolidone. Albendazole has been pro- the adults and 6 to 8% of the children in devel- posed as an alternative to metronidazole but is Despite the recognition of clinical illness in the G.lamblia has been reported to be highly last 40 years, there have been few reviews of susceptible to albendazole in vitro, but the therapy for this infection and no definitive ef- efficacy of drugs in clinical studies is fective treatment protocols have been pub- lished. In addition, only a handful of agents There is an obvious need for alternative an- which are available may have adverse effects or tigiardial agents. The aim of the present study be contraindicated in certain clinical situation. was to assess the effects of albendazole, met- Also, resistance may play a role in some infec- ronidazole against G.lamblia trophozoites in in *Corresponding author: Fax: +98-21-88013030,
E-mail: ghafarif@modares.ac.ir
bendazole were prepared in distilled water and Materials and Methods
dimethyl sulphoxide (DMSO) respectively. The final DMSO concentration in the culture tubes Isolation of G.lamblia cysts
was always <0.5%. Different concentrations of G.lamblia cysts were isolated from fresh feces each drug (0.05, 0.1, 2, 10, 50µg/ml) were pre- of patients with giardiasis in Tehran. The sam- ples showed no contamination to the other in- Trophozoites were exposed to different concen- testinal parasites and fungi. The average num- tration (0.05, 0.1, 2, 10, 50 µg/ml) of the drugs at 37 °C for 4 h. After chilling in ice water, tro- G.lamblia cysts were washed, purified and phozoites were counted by haemocytometer. In concentrated from feces using sucrose flotation control groups equivalent concentrations of dis- method with a simplified sucrose gradient tilled water and DMSO were used, in this re- method. The cysts after being washed twice in gard we used water as the control for metroni- distilled water were exposed with antibiotics and dazole group and DMSO (with the same con- fungicide at 4°C for a maximum of 4 days prior centration in stock solution) for albendazole group. The antiprotozoal activities of albenda- The excystation procedure was done using zole were compared with metronidazole within Bingham & Meyer technique (6). These procedures involved two steps: the induction of Statistical analysis
excystation performed in acid solution, and the The percentage of growth inhibition was calcu- culture and axenization in TYI-S-33 medium lated by comparison of growth rate of test supplemented with bile and heat inactivated group control group. The 50% inhibitory con- centration (IC50) was defined as the concentra- In excystation procedure 1 volume of clean cysts tion of the drug that inhibited growth by 50% as was added to 9 volumes of HCl in pH=2 and calculated by probit analysis. The 90% inhibitory 0.01N, and were incubated at 37 °C for 1h (5).
concentration (IC90) was similarly calculated (7). Culture and count
For this experiment we needed to prepare a large number of trophozoites. Hence the excysted parasites were added into the culture tubes con- From 37 human faecal samples containing G. taining 7 ml of TYI-S-33 (borosilicae glass Screw- lamblia cyst, 10 samples were excysted in axenic capped vials) and were incubated at 37 °C for 24-48 h. Trophozoites were harvested by chill- The IC50 calculated for each isolate after 4 h ing the tubes in ice water for 10-15 min. Then drug exposure, is shown in Table1. For met- counted by haemocytometer (Neubauer cell- ronidazole IC50 varied from 0.7 to 10 µg/ml counter chamber).The optimum trophozoite representing a range of variation of 14.28 fold concentration used was 50, 000 cells/ml (6). while IC90 varied from 6 to 42.5 µg/ml repre- Evaluation of parasite viability
senting a range of variation of 7.08 fold in sus- To assess trophozoites viability, eosine 0.1% ceptibility. For albendazole, the range of IC50 varied from 0.062 to 0.1µg/ml representing a Drugs assessment
range of variation of 1.61 fold while IC90 varied Metronidazole and albendazole were prepared from 1.5 to3µg/ml representing a range of vari- from Daru Pakhsh Co. The chemotherapeutic agents used were metronidazole and albenda- The results of IC50 in Table 1 showed that the zole. Stocks solution of metronidazole and al- mean for albendazole was 0.08 µg/ml and for metronidazole was 3.32 µg/ml. In this regard, Table 2 shows the comparison of percentage of the ratio of drug concentration was 3.32/0.08 killed trophozoites of G.lamblia following 4h (41.5) and showed albendazole than to metroni- exposure to different concentrations of met- Table 1: Susceptibility of G.lamblia isolates to (metronidazole and albendazole) in vitro condition.
Isolate No.
Metronidazole
Albendazole
Metronidazole
Albendazole
( µg/ml)
( µg/ml)
( µg/ml)
( µg/ml)
Table 2: Comparison of percentage of killed trophozoites of G.lamblia following 4h exposure to different concentration of
Drugs Drug
concentrations
Percentage of killed trophozoites
( µg/ml)
Following exposure to the drugs
Metronidazole
Albendazole
F Mohamadnezhad et al: In Vitro Effects of… Discussion
could be due to different strains of G.lamblia. The heterogeneity in drug sensitivity of parent The present study has demonstrated the supe- G.lamblia populations may be one of the fac- rior potency of albendazole against Giardia tors responsible for treatment failures of human trophozoites in vitro compared to metronida- zole. Our finding was similar to the other re- The results of this study are significant with ported data. Meloni et al. (1990) found that al- respect to prospects for a new approach to the bendazole was 5-10 times more active than me- tronidazole or tinidazole against G. lamblia as appears to be an ideal anti-giardial agent. It has judged the IC50 (8). Edlind et al. (1990) re- ported that albendazole was 50 times more ac- chemotherapeutic agents and is relatively tive than metronidazole (9). Upcroft et al. insoluble and poorly absorbed from the gut, (1999) reported that a great deal of variation in the antiprotozoal efficacies of the 13 com- parasites and should not affect the intestinal pounds tested was revealed. Only one com- pound was less effective than metronidazole against all three species of protozoa examined. Acknowledgements
All other compounds were as effective or more effective than metronidazole against some or all This study was supported by Tarbiat Modares organisms tested (10). According to Upcroft et University through pecuniary aid. The authors al. (2001) study the MIC for metronidazole wish to thank Mr. Poorghasem, Dr Farrahi from susceptible lines was 6.3 µM in those assays Danesh laboratory for kind cooperation and and that for the resistant lines was consistently Miss Ghasemi and Mrs Jaberi for kind help- higher (11). Majewska et al. (1991), found that ing.The authors declare that there is no Conflict all individual stocks were composed of parasite populations characterized by significantly (P<0.05) differing sensitivities to both ornida- References
zole and metronidazole (12). Farbey et al. (1995) reported that dose-response curves were Gardner TB, Hill DR. Treatment of Giardi- constructed for each isolate for metronidazole, the most common clinically used antigiardial agent, as well as for albendazole. Less than a 9- fold variation was found in the susceptibility of excystation and axenization of Giardia the isolates to albendazole, while for metroni- lamblia:in vitro susceptibility to metroni- dazole there was well over a 16,000 -fold varia- dazole and albendazole. Antimicrob Agents tion between the same groups of isolates (13). In 2003 clinical resistance against the drug has been reported by Wright et al., including cases where patients failed both metronidazole and bility of 11 clinical isolates of Giardia duodenalis from france. J Antimicrob Chem. usefulness of the existing drugs is the most cost-effective measure to ensure the continued Cedillo-Rivera R, Munoz O. In-vitro sus- availability of antigiardial drugs (14). ceptibility of Giardia lamblia to albenda- We found difference in activity of the drugs against various isolates. These differences peutic agents. J Med Microbiol. 199;37: 221-4. Iranian J Parasitol: Vol. 3, No. 2, 2008, pp. 38-42 J. In vitro excystation and subsequent ax- enic growth of Giardia lamblia. Trans R suscaptible and-resistant Giardia, Tricho- monas, and Entamoeba spp. Antimicrob SL. Production of viable Giardia cysts in 11. Upcroft JA, Upcroft P. Drug susceptibility vitro: determination by fluorogenic dye testing of anaerobic protozoa. Antimicrob staining, excystation, and animal infectiv- Gastroenterology. 1988;95:1–10. Giardia to metronidazole and ornidazole” JA, Seville P. Albendazole: a more effec- tive antigiardial agent in vitro than met- isolates of Giardia duodenalis from humans as assessed by an adhesion assay. Activity of the antihelminthic benzimida- 14. Wright JM, Dunn LA, Upcroft P, Upcroft zoles against Giardia lamblia in vitro. J JA. Efficacy of antigiardial drugs. Expert
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