Variability and variation in drug susceptibility among Giardia duodenalis isolates and clones exposed to 5-nitroimidazoles and benzimidazoles in vitro

Raúl Argüello-García, Maricela Cruz-Soto, Lydia Romero-Montoya and Guadalupe Ortega-Pierres*

Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados-IPN, Apartado Postal 14-740 07360 México, D.F., México

Received 10 December 2003; returned 10 April 2004; revised 21 May 2004; accepted 29 June 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives: We analysed, in a cell-by-cell study, the in vitro susceptibility of Giardia duodenalis strains, including Mexican isolates and their clones to 5-nitroimidazoles and benzimidazoles.

Methods: Fluorogenic dye staining (FDA-PI) and cell morphology (CM) assays, two fast and direct techniques, replaced the indirect ‘gold standard’ method (subculture in liquid medium) in the evaluation of 5-nitroimidazoles and benzimidazoles, respectively.

Results: Under these conditions, the activity of several 5-nitroimidazole and benzimidazole derivatives was consistent with their known efficacy, but parasite stocks exhibited a greater variability in response to 5-nitroimidazoles compared with benzimidazoles. Also, consecutive progenies from single stocks maintained in continuous subculture in drug-free media displayed changes (variations) in the proportions of drug resistant (R/T) subpopulations when exposed to sublethal concentrations of 5-nitroimidazoles and benzimidazoles. These were again more variable upon exposure to 5-nitroimidazoles than to benzimidazoles. Variations were not due to drug susceptibility shifts in parent trophozoites since analysis of cytokinetic processes showed a predominant pattern of susceptible/susceptible or resistant/resistant daughters, whereas susceptible/resistant daughters were scarce.

Conclusions: Our observations support the idea that G. duodenalis cultures exhibit variations in their response to 5-nitroimidazoles and benzimidazoles as a result of a drug-independent competition between drug-susceptible and drug-resistant subpopulations when parasites are subcultured.

Keywords: antiprotozoal drugs , clonal variation , viability assays , cell morphology assays , dye uptake assays


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Giardia duodenalis (syn. G. lamblia, G. intestinalis) infections are one of the most frequent waterborne causes of diarrhoea worldwide.1 Therapeutic strategy has included diverse pharmaceutical agents of traditional use such as metronidazole, quinacrine, furazolidone and paromomycin.2,3 Other drugs of more recent introduction, such as albendazole and nitazoxanide, have also been applied in clinical practice.4,5 Of these, metronidazole (a 5-nitroimidazole) and albendazole (a benzimidazole) may be considered the most representative anti-giardial agents of traditional and recent use, respectively. However, evidence points to an increasing frequency of cases refractory to treatment with these drugs,610 the causes of which include treatment non-compliance and emergence of drug-resistant Giardia.

In vitro evaluation of Giardia cultures is a useful way to assess the role of parasite variability in the outcome of treatment by chemotherapeutic agents. Studies on the comparison of sensitivity of G. duodenalis isolates and clones to metronidazole and albendazole have documented the presence of a heterogeneous pattern of response, a phenomenon termed variability or heterogeneity.1113 This is observed under equivalent drug-exposure conditions and by a variety of methodologies that measure cell viability by biochemical or physiological parameters in whole parasite populations.

In this context, experimental values for inhibitory concentrations at 50% (IC50) of metronidazole have been relatively similar when the same reference culture (PO/P1) was evaluated with distinct techniques, namely tritiated thymidine uptake (3H-TdR), in vitro adherence, subculture in liquid medium (SCLM) and nucleoside hydrolase activity, in different laboratories.1417 Nevertheless, in clones from this strain (e.g. P1C10) striking differences (15-fold) in IC50 values were observed when 3H-TdR and adherence assays were compared by the same group.18 Comparative studies with cultures derived from another reference strain (WB) have shown similar IC50 values for SCLM, morphology and oxygen uptake assays after a 3 h exposure to metronidazole.19 However, in more recent studies, SCLM and adherence assays after a 24 h exposure gave similar IC10 and IC50 values, but the corresponding IC90 determinations indicated a 2.6-fold difference for these methods.20 On the other hand, experimental determinations of the minimum lethal concentration (MLC) of albendazole in the P1 strain showed a 14-fold difference when in vitro adherence was used instead of SCLM,16,21 and a 30-fold difference in MLCs was obtained by in vitro adherence and 3H-TdR assays with the clone P1C10.18,21 Moreover, experimental values of IC50 for albendazole with the same strain (WB) and technique (growth inhibition assay) have shown a difference of almost two-fold.22,23

The differences observed in all these studies are most likely a consequence, at least in part, of using methods with distinct criteria and end points to evaluate trophozoite viability. However, the particular parasite culture used might itself play an active role in generating these apparently conflicting results. This idea is supported by the biological variation observed among Giardia stocks at several levels.24 In addition, most of the techniques used give indirect values because results are taken from comparisons of whole populations exposed, or not exposed, to drug. A finer analysis may be carried out by direct techniques in which each cell within a representative sample is evaluated, and thus the proportions of drug-resistant (R)/total (T) subpopulations is an efficient indicator of drug sensitivity. This study was performed to assess if Giardia cultures display not only variability in their response to 5-nitroimidazoles and benzimidazoles, but also if a single culture exhibits variation in its response to these drugs on the basis of a cell-by-cell analysis.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Parasite cultures and drugs

G. duodenalis trophozoites from reference strains P1 (ATCC # 30888) and WB (ATCC # 30957), Mexican isolates CIEA-0986–2 and CIEA-1286–1 obtained from acute symptomatic and asymptomatic patients, respectively,25 and isolates IMSS-0990–1 and IMSS-1090–1 obtained from Mexican patients with chronic asymptomatic infections26 were included in this study. Cloning of these stocks (strains and isolates) was performed by limiting dilution,27 and different numbers of clones were obtained from P1 (9), WB (14), CIEA-0986–2 (11), CIEA-1286–1 (11), IMSS-0990–1 (7) and IMSS-1090–1 (8). Culture was carried out in TYI-S-33 medium (ATCC 1404) supplemented with 0.05% bovine bile (Sigma)28 at 37°C, harvested by chilling in a water-ice bath and counted in a haemocytometer. Compounds of the 5-nitroimidazole group included metronidazole, tinidazole, ornidazole, dimetridazole, ronidazole (all from Sigma) and secnidazole (Rhone-Poulenc-Rorer, Mexico City), which were dissolved in sterile water. The benzimidazole derivatives used were albendazole, mebendazole, benzimidazole, oxibendazole, thiabendazole and nocodazole (Sigma) in N,N-dimethylformamide (DMF; Sigma) as solvent.

Time point evaluation of trophozoite viability after drug exposure

In all experiments, TYI-S-33 medium no older than 1 week and stored at 4°C was used to avoid cysteine oxidation and extrinsic variability in susceptibility to 5-nitroimidazoles.29,30 1 x 106 trophozoites from reference strains P1 and WB were exposed to different concentrations of metronidazole or albendazole for 24 h at 37°C in 4.5 mL screw-capped vials. After cell harvesting and counting, parasite viability was tested by the following methods:

  1. SCLM. This was performed as previously reported31 with standardized adaptations. 1 x 105 drug-exposed trophozoites were incubated in drug-free medium for 48 h at 37°C in 4.5 mL vials. After this, parasites were counted and the initial number of live (viable) trophozoites was extrapolated from a standard growth curve of non-drug-exposed parasites. The number obtained was divided by 1 x 105 to calculate the fraction of viable cells.
  2. 3H-TdR. The protocol reported by Boreham et al.14 was used with some standardized modifications. 1 x 106 drug-exposed parasites were pre-incubated at 37°C for 2 h in drug-free medium, then [3H]-thymidine (Amersham) with specific activity of 4.11 TBq/mmol was added at a concentration of 12.5 µCi/mL. After an additional 4 h incubation, parasites were washed 3–5 times with phosphate buffered saline (PBS) pH 7.2, and cell pellets were suspended in scintillation fluid to determine the amount of incorporated label in a Beckman 6000 liquid scintillation spectrometer. Trophozoite viability was estimated by regression analysis of the inhibition of 3H-TdR uptake by drug-exposed cultures.
  3. MTT reduction (MTT). The method of Wright et al.32 was followed with minor changes. Live Giardia trophozoites develop a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-formazan precipitate within cells stabilized by phenazine methosulphate (PMS; Sigma) and thus can be readily observed under light microscopy or quantified by spectrophotometry at 450 nm. For the purpose of this study, 1 x 106 PBS-washed, drug-exposed parasites were incubated for 45 min at room temperature in PBS containing 0.05% MTT (Sigma) and 0.2 mg/mL PMS. Cells were washed and observed under the microscope. At least 300 parasites were scored; those cells presenting brown MTT-formazan crystals were considered viable.
  4. Fluorogenic dye staining (FDA-PI). The method reported by Schupp & Erlandsen33 for Giardia cysts was adapted and standardized for trophozoites in this study. 1x106 PBS-washed, drug-exposed parasites were incubated for 5 min at 37°C in a mixture of 100 µL of 40 µg/mL fluorescein diacetate (FDA; Sigma) in PBS and 100 µL of 30 µg/mL propidium iodide (PI; Sigma) in PBS. Samples were mounted on coverslips and parasites observed under white light and under a blue 450–490 FT510 LP520 filter in a Zeiss epifluorescence photomicroscope—model MC 80—fitted with UV light. Trophozoites displaying a brilliant green fluorescence—associated with the accumulation of fluorescein as a product of esterase-mediated hydrolysis of FDA—were scored as viable. Parasites with disrupted membranes displayed a brilliant red–orange fluorescence due to PI incorporation, associated or not with nucleic acids, and were scored as non viable.34 Cell counts were based on at least 300 parasites per sample.
  5. Cell morphology (CM). Drug-exposed parasites used for SCLM were also evaluated by this technique. Parasite pellets were washed three times with TYI-S-33 medium to remove any remaining drug before observation at 400x magnification. Deformations in typical trophozoite appearance, such as the presence of a distorted membrane contour, an irregular cell shape, a significantly increased size and abnormally granular cells were included as criteria for scoring non-viable parasites. Cell counts were based on at least 300 trophozoites per sample.

Longitudinal analysis of drug susceptibility

Parasites from stocks and clones were maintained in continuous subculture and passaged 2–3 times a week depending on the culture. Passage was carried out when the culture reached 100% confluence in 15 mL conical bottom tubes (Falcon), corresponding to log phase of growth. At this time, flasks were chilled on ice for at least 20 min and detached parasites harvested by manual agitation and decantation of cold medium. Fresh medium was added to refill flasks and these were incubated again at 37°C until complete confluence was reached. Trophozoites obtained at each harvest were counted, exposed to drugs as indicated and cell viability was determined by the method selected as optimum in each case.

Statistical analysis and definitions

In time point assays of drug susceptibility, IC50 and MLC values of 5-nitroimidazoles and benzimidazoles are defined as the concentrations of drug in which 50% or no viable Giardia trophozoites, respectively, were observed by the criterion of viability used in each method. These were calculated from dose-response curves by regression analysis (least square method) and the 95% confidence limits (CL95) were calculated from the plot of the logit value against the logarithm of drug concentration. Comparisons of values for these parameters were carried out by Student's t-test. In longitudinal analyses, analysis of variance (ANOVA) (time/progenies and drug as factors) was used. For time point and longitudinal analyses, the proportions of drug-resistant subpopulations (R/T) are defined as the fraction of viable cells within a whole population in which non-viable cells are the supplementary fraction, as calculated by MTT, FDA-PI and CM assays.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Determination of viability in drug-exposed trophozoites by different methods

P1 and WB strains of G. duodenalis exposed to metronidazole or albendazole and evaluated by SCLM, FDA-PI, 3H-TdR and MTT assays showed different dose-response patterns depending on the method used to determine cell viability. SCLM yielded the lowest MLCs. These were 5.9 µM and 1.6 µM for metronidazole on WB and P1, respectively (Table 1). Taking these latter as reference, exposure to metronidazole showed MLC values 10- and 38-fold greater as determined by FDA-PI, 16- and 39-fold greater as determined by 3H-TdR, whereas MLC values were 20- and 119-fold greater in MTT assays for strains WB and P1, respectively. In spite of the 10–38-fold difference between SCLM and FDA-PI, this latter was acceptable for our purposes since: (a) the use of FDA-PI for 5-nitroimidazole comparisons in structure-activity assays (see next section) gave qualitatively similar results to SCLM (data not shown), i.e. determinations are distinct only in a sense of magnitude and (b) FDA-PI has several advantages in terms of cost, performance time and possible automization. FDA-PI, which is a direct, easier and faster cell-by-cell assay was used for the remainder of this study instead of SCLM to evaluate the effect of 5-nitroimidazoles on Giardia viability.


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Table 1. Determinations of IC50 and MLC values for G. duodenalis trophozoites of WB and P1 strains exposed in vitro to metronidazole or albendazole by different methods. In all cases, the value of the mean (drug concentration in µM) obtained in four independent experiments performed in duplicate is shown, and the 95% confidence limits (in parentheses) are indicated in each case

 
On the other hand, exposure to albendazole showed greater differences in MLC values obtained by FDA-PI (463.1 µM and 1500 µM for WB and P1, respectively) when compared with the other techniques (Table 1). MLCs determined by FDA-PI were 950- and 1691-fold greater, MLC values determined by 3H-TdR were 3603- and 1761-fold greater, and MTT assays gave MLCs 3648- and 2291-fold greater for WB and P1, respectively. None of the techniques used substituted for SCLM to evaluate the effect of benzimidazoles without a >1000-fold decrease in sensitivity. In general, MLC values for drugs against WB and P1 strains were quantitatively different but qualitatively similar as determined in different assays (Table 1).

CM assays were not used with metronidazole because this drug did not exert a consistent effect on trophozoite morphology at concentrations (0.3–1000 µM) that proved effective by other methods used. In contrast, albendazole induced dramatic changes in trophozoite morphology at concentrations within effective ranges (0.1–1.0 µM) as assessed by microscope observation. This agrees with previous observations22 and raised the hypothesis that once a trophozoite is damaged by albendazole at the morphological level, it is not longer able to replicate. Indeed, in separate experiments cell replication rates by inocula with increasing proportions of trophozoites morphologically altered by exposure to several albendazole concentrations were analysed (Figure 1). Two types of inocula were used: (a) increasing numbers of drug-exposed trophozoites in which the proportions of deformed and non-deformed cells were determined by CM analysis, or (b) increasing numbers of untreated, non-deformed trophozoites in an inoculum the size of which was equal to the corresponding number of non-deformed cells quantified as described in (a), bearing in mind each albendazole concentration and inoculum size. Thus in both cases the initial number of non-deformed cells was the same. As shown in Figure 1, cell replication was progressively impaired when parasites were treated with increasing albendazole concentrations (0.12–0.45 µM) (P < 0.001), whereas inoculum size did not influence cell growth rate in all cases except under high albendazole concentrations (0.45 µM, P = 0.049). Drug-treated cells showed a lower growth rate than the untreated counterpart. This is consistent with the expected response, rendering CM a criterion with equal or higher sensitivity than cell replication as determined by SCLM. In a representative comparison, IC50 and MLC values obtained by SCLM (see Table 1) and CM [IC50 and (CL95) of 0.08 µM (0.06, 0.11); MLC and (CL95) of 0.16 µM (0.13, 0.21)] in the WB strain did not show a significant difference between these values (P > 0.05). Therefore we used CM assay as a direct, fast and very sensitive cell-by-cell method to substitute for SCLM in evaluating the effect of benzimidazoles in this study.



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Figure 1. Comparison of cell growth in albendazole-exposed parasites displaying or not deformations in cell shape. Increasing numbers of G. duodenalis trophozoites of WB strain (x-axis) were incubated for 48 h at 37°C and cell counts were determined (y-axis). Two types of inoculum were used: drug-exposed trophozoites in which the proportion of deformed cells was quantified by CM analysis (open circles) and non-treated trophozoites (crosses). In both cases, the total number of non-deformed cells in the inoculum was the same. A curve without cell growth is included (open squares). Albendazole concentrations used were: (a) 0.12 µM, (b) 0.30 µM, (c) 0.35 µM and (d) 0.45 µM. Each point corresponds to mean values of four independent experiments performed in duplicate. For a better appreciation of the data, standard deviations are not included but these were generally <0.15 of the value of the corresponding mean.

 
Comparison of the efficacy of different 5-nitroimidazole and benzimidazole compounds

Experimental dose-response curves for each compound were obtained and the corresponding mean MLC values from four independent experiments were calculated. The cell-by-cell determination by FDA-PI assays of the relative efficacy of six 5-nitroimidazoles showed tinidazole and ornidazole as the most active compounds [MLC and (CL95): 5.8 µM (4.9, 6.5) and 9.1 µM (8.0, 9.8), respectively] whereas dimetridazole, metronidazole and secnidazole exhibited a lower efficacy [MLC and (CL95): 65.0 µM (57.9, 71.3), 59.3 µM (51.7, 69.2) and 83.1 µM (69.5, 93.6), respectively]. Ronidazole, a compound the chemical structure of which appears to be ‘hybrid’ between dimetridazole and albendazole with a non-methyl group in position 2, had an intermediate efficacy [MLC and (CL95): 23.9 µM (18.9, 29.1)] (Figure 2a).



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Figure 2. Dose-response curves of 5-nitroimidazoles (a) and benzimidazoles (b). G. duodenalis trophozoites were exposed to increasing concentrations of the indicated compounds for 24 h at 37°C, and cell viability was determined by FDA-PI staining (a) and by CM assay (b). Each point corresponds to mean values of four independent experiments performed in duplicate. For a better appreciation, standard deviations are not included but these were generally <0.15 of the value of the corresponding mean.

 
Among six benzimidazoles tested cell-by-cell in CM assays, the four benzimidazole 2-carbamates used (i.e. nocodazole, albendazole, mebendazole and oxibendazole) exhibited high efficacy [MLC and (CL95): 0.08 µM (0.06, 0.10), 0.16 µM (0.13, 0.21), 0.17 µM (0.13, 0.20) and 0.36 µM (0.29, 0.46), respectively]; thiabendazole had an intermediate efficacy [MLC and (CL95): 25.0 µM (18.8, 31.4)] and benzimidazole, as expected from its lack of active groups, displayed poor activity [MLC and (CL95): 9.1 mM (6.9, 13.8)] (Figure 2b).

When metronidazole and albendazole where taken as reference for statistical comparison, all 5-nitroimidazoles—except for dimetridazole (P < 0.05)—showed a very distinct structure–activity pattern (P < 0.005). Among benzimidazoles, mebendazole displayed similar activity to albendazole, whereas remaining drugs showed a different pattern (P≤0.01). From these compounds we selected four representative drugs (metronidazole, tinidazole, albendazole and mebendazole) for further evaluations on the basis of their proven high in vitro efficacy and wide use in clinical giardiasis.

Time point evaluations of drug sensitivity of Giardia stocks and clones

IC50 concentrations of metronidazole, tinidazole, albendazole and mebendazole were experimentally determined for a reference strain (WB) by FDA-PI or CM assays. These concentrations were used to evaluate all remainder stocks and clones, and proportions of R/T were obtained (Table 2). Cultures displaying fractions of viable cells (R/T) <0.25 were considered susceptible, those with an R/T index between 0.26 and 0.75 were considered as intermediate susceptibility and cultures with R/T values >0.75 were considered tolerant. All 11 clones derived from isolate CIEA-0986–2 were tolerant to both 5-nitroimidazoles and benzimidazoles, whereas striking differences among clones of the five remaining stocks were observed (Table 2). Independently from Giardia stocks, clones exposed to albendazole and mebendazole exhibited a much more restricted susceptibility pattern (i.e. lower heterogeneity) than when exposed to metronidazole and tinidazole. With the exception of stock CIEA-0986–2, clones from all remaining stocks displayed striking variations in their rates of R/T (Table 2). For instance, the WB strain from which the experimental IC50s were used, in spite of displaying intermediate susceptibility to albendazole (R/T, 0.64) and mebendazole (R/T, 0.56), as expected from previous evaluations, now exhibited a pattern of metronidazole tolerance (0.83) and tinidazole susceptibility (R/T, 0.03). Its clones WB-1 and WB-12, on the other hand, were qualitatively similar, but the former was metronidazole susceptible (R/T, 0.02) and the latter albendazole susceptible (R/T, 0.00). IMSS-1090–1, a multitolerant isolate, gave rise to a clone (IMSS-1090–1–7) that was multitolerant as well, but metronidazole susceptible.


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Table 2. Time point analysis of in vitro susceptibility of G. duodenalis isolates and clones to selected 5-nitroimidazoles and benzimidazoles. Cell viability is expressed as the fraction of drug-resistant parasites/total parasites (R/T) for each culture separately exposed for 24 h at 37°C to tinidazole or metronidazole by using FDA-PI staining, and to albendazole or mebendazole by using CM assays. R/T values correspond to determinations in at least 300 cells from each culture sample

 
Longitudinal changes in drug susceptibility

The possibility that individual stocks or clones might display significant changes in their pattern of drug susceptibility, if maintained in continuous subculture, was assessed by longitudinal analyses, and representative results from one stock and three clones are shown in Figure 3. Not surprisingly, all cultures showed constant values of R/T when successive progenies obtained from the same culture recipient were exposed to mebendazole. Exposure to albendazole, however, induced recurrent changes in cell viability, but the most frequent change was from tolerance to intermediate susceptibility or vice-versa; changes in tolerance-susceptibility were occasional in some cultures (e.g. P1, WB-1, IMSS-0990–1–2 and IMSS-1090–1–7) (Figure 3). The R/T data were clearly more variant when successive progenies were exposed to either metronidazole or tinidazole and thus tolerance-susceptibility turnovers were much more frequent (compare all graphs in Figure 3). Interestingly, an apparent cyclic pattern of susceptibility change was observed in some cultures (WB-1, P1, and IMSS-1090–1–7) after tinidazole exposure similar to that seen for IMSS-0990–1–2 (Figure 3). Other cultures with this latter pattern were WB, WB-12, IMSS-0990–1 and IMSS-0990–1–7. Metronidazole exposure-induced changes were generally more erratic for all stocks and clones tested. WB strain was the unique culture that behaved relatively constantly upon metronidazole exposure when tested over 40 days (not shown). Statistical analysis suggested that the drug to which all parasites were exposed influenced the susceptibility pattern (P < 0.05) and that in all except for some cultures (WB1 and P1, P > 0.4) the analysed progenies showed this variant pattern (P < 0.05).



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Figure 3. Representative longitudinal analysis of in vitro susceptibility of G. duodenalis isolates and clones to selected 5-nitroimidazoles and benzimidazoles. Cell viability [expressed as the fraction of drug-resistant parasites/total parasites (R/T)] was determined in duplicate samples of consecutive progenies from each culture maintained under continuous subculture and exposed for 24 h at 37°C to IC50 of tinidazole (closed triangles) or metronidazole (open diamonds) by using FDA-PI staining, and to IC50 of albendazole (closed circles) or mebendazole (open squares) by using CM assays. Values in the graph correspond to the mean of determinations in at least 300 cells for each time point indicated.

 
Origin of variation in drug susceptibility

To assess the most likely origin of this variation in drug susceptibility, cell viability of dividing cells was scored in cultures exposed to metronidazole and albendazole at their corresponding IC50 obtained by FDA-PI and CM assays, respectively. Figure 4 shows microscope observations of mitotic events in cultures of WB trophozoites immediately following exposure to metronidazole or albendazole. In both cases, daughter cells were determined to be both viable, both non-viable or one viable and the other non-viable, being the latter pattern only occasionally (<2%) and the other two of higher frequency (30–60%). Therefore, in a given culture, most daughter cells originated from trophozoites with similar patterns of drug susceptibility (either resistant or sensitive).



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Figure 4. Microscope analysis of the pattern of resistance/susceptibility in dividing trophozoites exposed to metronidazole (A–F) or albendazole (G–I). Parasite cultures of WB strain were exposed for 24 h at 37°C to IC50 doses of metronidazole or albendazole, and cell viability assays (FDA-PI staining and CM respectively) were performed. Cells undergoing cytokinesis were observed under phase contrast (A–C, G–I) and under fluorescence microscopy (D–F). Panels A–C show mitotic processes after metronidazole exposure in which both cells are viable (D), one is viable and the other non-viable (E) or both are non-viable (F). In G, a mitosis with two daughter cells without morphological damage is shown, whereas in H one of the cells displays morphological alterations, and in I both cells are deformed.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
G. duodenalis viability after drug exposure has been evaluated in vitro by several criteria, including physiological (adhesion to both plastic walls and epithelial cells, motility, growth)13,35,31 and biochemical (enzyme activities, oxygen consumption, incorporation of radiolabelled precursors)32,17,19,36 parameters. We have shown here that morphology is particularly useful to evaluate the effect of benzimidazoles. Biochemical assays have been used to evaluate 5-nitroimidazoles but none is as sensitive as SCLM. The usefulness of these parameters reflects the intracellular pathways of damage by benzimidazoles (ß-tubulin binding) and 5-nitroimidazoles (generation of cytotoxic radicals). The majority of evaluation protocols reported in the literature are indirect measurements of drug activity because parasite populations have been analysed as a whole. The use of protocols measuring effects directly on single cells may reduce the time taken for evaluation and give in vitro results with a qualitative relationship to in vivo observations from clinical and experimental infections.

Our data showed that SCLM is a highly stringent and sensitive method for assessing the effects of 5-nitroimidazoles and benzimidazoles (i.e. a ‘gold standard’). However, it is an indirect and time-consuming assay that was efficiently replaced by the direct assays described here, without significantly affecting efficacy or quality of data. Thus, dye uptake and morphological changes were sensitive and direct measurements of cell viability when Giardia trophozoites were exposed to 5-nitroimidazoles, as metronidazole and benzimidazoles as albendazole, respectively. In addition, it was emphasized that the selection of the best direct method to determine Giardia viability was highly influenced by the modes of action of 5-nitroimidazoles and benzimidazoles, since fluorogenic dye staining (FDA-PI, biochemical) and cell morphology (CM, physiological) assays were optimal. These are fast, low-cost and efficient techniques that also offer the possibility of automization if a flow cytometer is available and large quantities of samples have to be evaluated. For instance, other protozoa have been analysed by FDA-PI staining coupled to automated counting by flow cytometry to determine the effect of cytotoxic agents on the Trichomonas vaginalis membrane37 or to detect programmed cell death in Blastocystis hominis.38 Equivalent protocols with calcein-AM and ethidium bromide have also been reported for Tetrahymena piriformis.39

In addition, data from these cell-by-cell protocols in time point assays are validated by their close correlation with other in vitro evaluations and with in vivo observations in experimental and clinical giardiasis. This in vitro–in vivo correlation has been previously assessed with indirect methods and animal models. For several 5-nitroimidazoles included in our work (metronidazole, tinidazole, ornidazole, ronidazole and secnidazole), data from the neonatal mouse model correlated with that of the 3H-TdR uptake assay.40 Albendazole efficacy was also confirmed in the mouse model, using adhesion and regrowth assays as reference.21,41 Our time point evaluations of drug susceptibility also showed tinidazole to be a 5-nitroimidazole of high efficacy. Taking the MLC of tinidazole as a reference (i.e. with an arbitrary index of 1.0), the relative efficacy calculated for ornidazole (0.6), secnidazole (0.1) and metronidazole (0.1) correlates well with their in vivo activity in the neonatal mouse model (see Table 2 of ref. 40). This extrapolation is supported by the fact that, as shown in Table 1, the 3H-TdR uptake assay was a method of comparable sensitivity to FDA-PI staining. Ronidazole, a ‘hybrid’ compound bearing two all-important groups, one of the benzimidazoles and one of the 5-nitroimidazoles (2-carbamate and 5-nitro, respectively) was more effective than metronidazole and, given its favourable therapeutic profile,40 further evaluations of this compound should be carried out.

As far as the patterns of drug susceptibility in G. duodenalis are concerned, our data with direct protocols confirmed that this parasite displays a broad variability in response to drugs of clinical use, including metronidazole and albendazole.1113,42 The molecular basis of this feature and its causes are still undefined. An interesting observation is that in vitro exposure to sublethal doses of metronidazole can prevent the competitive exclusion between mixed Giardia cultures with distinct growth rates, which is observed in the absence of the drug.43 In a therapeutic sense, it is assumed that the repetitive use of suboptimal doses of a drug (either a 5-nitroimidazole or a benzimidazole) may also lead to the predominance of drug-tolerant populations and thus to therapy failure by the parasite's drug resistance. This possibility is supported by clinical and experimental data: (a) communities receiving antihelmintic treatments with mebendazole, which use suboptimal doses for giardiasis, may exhibit subsequent increasing rates of infections by Giardia,44 and (b) exposure to sublethal doses of drug is an effective method of obtaining drug-resistant cultures in vitro.45 Thus, in the present study, we used suboptimal drug concentrations (IC50) in particular cultures to obtain additional insights into the characterization of drug susceptibility/resistance in this parasite.

By using IC50 concentrations of representative 5-nitroimidazoles (metronidazole and tinidazole) and benzimidazoles (albendazole and mebendazole) in longitudinal studies (up to 40 days) of drug susceptibility with direct methods, our observations suggest that Giardia is able to display not only variability but variation in the response of an individual culture to drugs. This variation is spontaneous and, independent of the method used to determine cell viability, is demonstrated when sublethal doses of the drug are used in the evaluation of progenies (Figure 3). In similar experiments using successive progenies of WB strain and exposing them to MLCs of metronidazole and albendazole, no significant changes in drug susceptibility were observed (data not shown). This is consistent with the advantage of using IC50 to characterize variation in drug susceptibility in this parasite, and the use of MLC as a more reliable breakpoint to define susceptible and resistant populations of microorganisms,46 and should be also convenient to compare drug susceptibility assays. This is supported by other studies where IC50-based estimations cannot distinguish the relative efficacy of methods such as adhesion and SCLM for metronidazole, in which the latter is more sensitive in an MLC-based comparison.20 A similar observation was obtained by comparing FDA-PI and 3H-TdR assays for metronidazole and MTT and 3H-TdR assays for albendazole in the WB strain (Table 1).

Variation in drug susceptibility could be a process limited in terms of the range of drug concentration in which it occurs, but seems to be common for most Giardia stocks. The parasite genotype might be irrelevant in this context because all of our Mexican isolates used in this study are of the AI subtype.26 Related factors could be the spontaneous changes observed in the expression of variable surface proteins (VSPs) in this parasite.47 A correlation between the expression of RNAs for certain VSPs and a variant resistance to albendazole is likely to occur and its analysis is in progress in our laboratory. On the other hand, this variation does not come from a possible shift of drug susceptibility in parental cells since the analysis of mitotic processes showed that drug-tolerant or drug-susceptible daughter trophozoites originated from similar preceding parasites, suggesting that drug-resistant parasites could arise as a predominant subpopulation only from a selective process given by the conditions in which the parasite is actually growing.

In conclusion, our data emphasize the biological plasticity of G. duodenalis and the need to couple the clinical use of a defined chemotherapeutic agent with an adequate method to determine susceptibility and resistance of the parasite in epidemiological studies. Also, these observations might help to explain the diversity in drug susceptibilities of Giardia reported in the literature.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We are grateful to Derek Wakelin and Jacqueline Upcroft for critically reading this manuscript. We also thank Leticia Juárez Salas for secretarial assistance, René López Bolaños and Blanca Herrera Ramírez for technical assistance, and Arturo PérezTaylor Reyes for part of the art work. This work was partially supported by a grant from CONACyT-Mexico (ref. 3751P-M9607).


    Footnotes
 
* Corresponding author: Tel: +525-50613800 ext. 5331; Fax: +525-50617100; Email: gortega{at}mail.cinvestav.mx


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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