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
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Abstract |
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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
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Introduction |
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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.
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Materials and methods |
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G. duodenalis trophozoites from reference strains P1 (ATCC # 30888) and WB (ATCC # 30957), Mexican isolates CIEA-09862 and CIEA-12861 obtained from acute symptomatic and asymptomatic patients, respectively,25 and isolates IMSS-09901 and IMSS-10901 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-09862 (11), CIEA-12861 (11), IMSS-09901 (7) and IMSS-10901 (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:
Longitudinal analysis of drug susceptibility
Parasites from stocks and clones were maintained in continuous subculture and passaged 23 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.
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Results |
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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 1038-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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CM assays were not used with metronidazole because this drug did not exert a consistent effect on trophozoite morphology at concentrations (0.31000 µM) that proved effective by other methods used. In contrast, albendazole induced dramatic changes in trophozoite morphology at concentrations within effective ranges (0.11.0 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 (0.120.45 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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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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When metronidazole and albendazole where taken as reference for statistical comparison, all 5-nitroimidazolesexcept for dimetridazole (P < 0.05)showed a very distinct structureactivity pattern (P < 0.005). Among benzimidazoles, mebendazole displayed similar activity to albendazole, whereas remaining drugs showed a different pattern (P0.01). 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-09862 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-09862, 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-10901, a multitolerant isolate, gave rise to a clone (IMSS-109017) that was multitolerant as well, but metronidazole susceptible.
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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-099012 and IMSS-109017) (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-109017) after tinidazole exposure similar to that seen for IMSS-099012 (Figure 3). Other cultures with this latter pattern were WB, WB-12, IMSS-09901 and IMSS-099017. 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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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 (3060%). 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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Discussion |
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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 vitroin 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.
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Acknowledgements |
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Footnotes |
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References |
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