Journal of Antimicrobial Chemotherapy (2000) 46, 819-821
© 2000 The British Society for Antimicrobial Chemotherapy
Brief report |
Metronidazole and albendazole susceptibility of 11 clinical isolates of Giardia duodenalis from France
a Laboratoire de Parasitologie Expérimentale, CHU Charles-Nicolle, 76031 Rouen Cedex; b Laboratoire de Parasitologie, CHU Amiens-Sûd, 80054 Amiens Cedex 1; c Laboratoire de Parasitologie, Hôpital Edouard-Herriot, 8 avenue Rockfeller, 69373 Lyon; d Laboratoire d'Immunologie et d'Immunopathologie, CHU Clémenceau, 14033 Caen, France
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Abstract |
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The metronidazole and albendazole susceptibility of 11 clinical isolates of Giardia duodenalis from France was determined using a neonatal mouse model and compared with the outcome in patients after standard metronidazole therapy (0.75 g/day for 5 days). All isolates found to be clinically resistant to metronidazole (4/11) exhibited an ID50 > 120 mg/kg in the mouse model. This therefore appears to be a suitable animal model in which to explore drug failures in human giardiasis.
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Introduction |
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In human giardiasis, therapeutic failure is occurring more and more frequently, due to low compliance with drug therapy, reinfestation or parasite resistance to metronidazole and/ or the nitroimidazole-related compounds secnidazole, tinidazole and ornidazole.1 In such cases, albendazole has been proposed as an alternative to metronidazole but is not always effective. The aim of this study was to evaluate the use of a neonatal mouse model to establish the cause of therapeutic failures.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Giardia isolates
Faecal samples containing Giardia cysts were obtained from three French University Hospitals over a 6 month period (Amiens University Hospital, 13 samples; Lyon University Hospital Edouard Herriot, seven samples and Rouen University hospital, 10 samples). Patients received standard metronidazole therapy (0.75 g/day for 5 days) (with the exception of two HIV-infected patients who received 1.5 g/day for 7 days) and the outcome of treatment was noted. If treatment failure occurred, patients received a standard albendazole therapy (0.4 g/day for 5 days). One patient dropped out of the study.
Faeces were diluted in water and cysts were isolated using sucrose gradients.2 Parasites were stored in liquid nitrogen.3
Giardia excystation in gerbils
All animals were managed according to the regulations of the French Ministry for Agriculture. Mongolian gerbils (Meriones inguiculatus) were used as the animal model for Giardia excystation.4 Briefly, animals were killed 7 days after oral infection (104–105 cysts/animal) and the intestinal contents were examined for the presence of trophozoites. This procedure resulted in an apparent excystation in 11 of 30 isolates that were used in the present work. Failure was due to parasite death [as identified by means of propidium iodide (PI) staining] in six out of 19 isolates but the cause of failure was not established in the remainder.
Evaluation of drug susceptibility in a mouse model
Drugs were purchased as pure powders (Sigma, San Quentin Fallavier, France). Drug susceptibility was determined using the NMRI neonatal mouse model described previously.5,6 Briefly, 6-day-old suckling mice were infected with 105 trophozoites in 100 µL of MHSP3 medium via an intragastic animal feeding biomedical needle (Poppers and Sons, Inc., New York, NY, USA) attached to a 1 mL syringe.7 On day 6 post-infection, half of each litter was treated by gavage with 100 µL of albendazole dispersed in water containing 0.5% of methyl cellulose, or metronidazole diluted in a 0.9% NaCl solution in water. The other half of the litter served as the control and received methyl cellulose or NaCl alone. Forty eight hours after treatment (i.e. day 8 post-infection) the mice were killed and the entire small intestine was removed, opened longitudinally and placed in 3 mL cold NCTC 135 medium (Life Technologies, Cergy-Pontoise, France) for at least 10 min. Tubes were vortexed to ensure complete detachment of parasites. Giardia trophozoites were separated from the mucosa by gauze filtration and filtrates were mixed with 100 µL of an aqueous solution of formaldehyde. Parasites were counted in a haemocytometer. For each isolate, four doses (10, 50, 100 and 200 mg/kg) of each drug were tested twice, using one litter for each dose (i.e. a total of 12–32 animals/ dose).
Expression of results
A log transformation was applied to the percentage of surviving trophozoites and the dose required to eliminate 50% of the trophozoites (ID50) was computed from a plot value against the logarithm of drug concentration. From these data, it was observed that one standard deviation accounted for less than 10% of the mean value.
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Results |
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As shown in the Table
, isolates were distributed in three groups according to their ID50 of metronidazole: three isolates with ID50 from 31.0 to 32.9 mg/kg, three isolates with ID50 from 71.5 to 81.5 mg/kg and five with ID50 > 120 mg/kg. In this latter group, three out of five isolates were found to be clinically resistant to standard metronidazole therapy. Figures 1 and 2 illustrate dose–response examples for one drug-sensitive and one drug-resistant Giardia isolate. In one of the two HIV-infected patients, treatment with metronidazole 1.5 g/day for 7 days was successful. With albendazole, the isolates were distributed in three groups: five isolates with ID50 < 10 mg/kg, 5 with ID50 from 30 to 53 mg/kg and one very resistant isolate in which the ID50 was 161 mg/kg.
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) and one albendazole-resistant (Rouen/99/lpe/6, 
) Giardia isolate. Rouen/99/lpe/3: y = –1.631 x +6.761; Rouen/ 99/lpe/6: y = –0.271x +5.29.
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Clinically, 7/11 isolates responded to metronidazole therapy (0.75 g/day for 5 days for six patients and 1.5 g/day for 7 days for one patient), one was clinically resistant to metronidazole (0.75g/day for 5 days) but responded to albendazole (0.4 g/day for 5 days) while two isolates were clinically resistant to both drugs: metronidazole 0.75 g/day for 5 days for one patient, 1.5 g/day 7 days for the other patient and albendazole 0.4 g/day for 5 days for both (Table
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Discussion |
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ID50 values obtained for metronidazole-sensitive isolates in this study were similar to those reported previously.8 All isolates associated with metronidazole treatment failure exhibited an ID50 greater than 120 mg/kg, which suggests that an ID50 breakpoint of 120 mg/kg may be useful in distinguishing drug resistance as a cause of treatment failure from other causes. In this context, the metronidazole treatment failure that occurred in this study appears to be due to true drug resistance. Interestingly, the patient infected by the isolate Rouen/98/lpe/9 (ID50 150 mg/kg) was cured by a higher dose of metronidazole (1.5 g/day for 7 days instead of 0.75 g/day for 5 days) whereas the patient infected by isolate Rouen/98/lpe/11 (ID50 = 125.2 mg/kg) was not. An increase in drug dose regimen may therefore be helpful in the management of some cases of clinically resistant human giardiasis.
A standard albendazole therapy was used to treat only the three patients infected with isolates that were resistant to metronidazole. Two of these isolates, which were less sensitive to albendazole in the neonatal mouse model, were clinically resistant to albendazole (0.4 g/day for 5 days). This is in agreement with a previous clinical study that reported treatment failures with both metronidazole and albendazole.9 In spite of the difficulties linked to the use of experimental animals, our results demonstrate the value of a neonatal mouse model to explore drug failures in human giardiasis.
Clinically, therapeutic failure with standard metronidazole therapy occurred in three of 11 patients. Although the high number of Giardia metronidazole-resistant strains found in this study may be explained in part by the referral of patients with therapeutic failure to the University hospital, clinical resistance to metronidazole and/or to albendazole does not appear to be uncommon.9 This underlines the need for new antigiardial drugs such as lactone-substituted imidazoles or nitazoxanide.10,11
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Acknowledgments |
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This work was supported in part by a grant from the Ministère Français de l'Environnement, 1998.
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Notes |
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* Corresponding author. Tel/Fax: +33-2-35-14-85-81; E-mail: llemee1234{at}aol.com
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References |
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1 . Townson, S. M., Boreham, P. F., Upcroft, P. & Upcroft, J. A. (1994). Resistance to the nitroheterocyclic drugs. Acta Tropica 56, 173–94.[Web of Science][Medline]
2
.
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, 4610–3.
3 . Phillips, R. E., Boreham, P. F. & Shepherd, R. W. (1984). Cryopreservation of viable Giardia intestinalis trophozoites. Transactions of the Royal Society of Tropical Medicine and Hygiene 78, 604–6.[Web of Science][Medline]
4
.
Isaac-Renton, J. L., Shahriari, H. & Bowie, W. R. (1992). Comparison of an in vitro method and an in vivo method of Giardia excystation. Applied and Environmental Microbiology 58, 1530–3.
5
.
Boreham, P. F., Phillips, R. E. & Shepherd, R. W. (1986). The activity of drugs against Giardia intestinalis in neonatal mice. Journal of Antimicrobial Chemotherapy 18, 393–8.
6 . Chochillon, C., Favennec, L., Gobert, J. G. & Savel, J. (1990). Giardia intestinalis: study of infestation procedures of young mice before weaning. Comptes Rendus des Seances de la Societe de Biologie et de ses Filiales 184, 150–7.[Web of Science][Medline]
7 . Favennec, L., Cochillon, C., Meillet, D., Magne, D., Savel, J., Raichvarg, D. & Gobert, J. G. (1990). Adherence and multiplication of Giardia intestinalis on human enterocyte-like differentiated cells in vitro. Parasitology Research 76, 581–4.[Web of Science][Medline]
8 . Boreham, P. F. & Upcroft, J. A. (1991). The activity of azithromycin against stocks of Giardia intestinalis in vitro and in vivo. Transactions of the Royal Society of Tropical Medicine and Hygiene 85, 620–1.[Web of Science][Medline]
9 . Brasseur, P. & Favennec, L. (1995). Two cases of giardiasis unsuccessfully treated by albendazole. Parasite 2, 422.[Web of Science][Medline]
10 . Romero Cabello, R., Guerrero, L. R., Munoz Garcia, M. R. & Geyne Cruz, A. (1997). Nitazoxanide for the treatment of intestinal protozoan and helminthic infections in Mexico. Transactions of the Royal Society of Tropical Medicine and Hygiene 91, 701–3.[Web of Science][Medline]
11
.
Upcroft, J. A., Campbell, R. W., Benakli, K., Upcroft, P. & Vanelle, P. (1999). Efficacy of new 5-nitroimidazoles against metronidazole-susceptible and -resistant Giardia, Trichomonas, and Entamoeba spp. Antimicrobial Agents and Chemotherapy 43, 73–6.
Received 11 October 1999; returned 8 December 1999; revised 7 February 2000; accepted 14 July 2000
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