A novel Giardia lamblia nitroreductase, GlNR1, interacts with nitazoxanide and other thiazolides

The nitrothiazole analogue nitazoxanide [NTZ; 2-acetolyloxy-N-(5-nitro-2-thiazolyl)benzamide] represents the parent compound of a class of drugs referred to as thiazolides and exhibits a broad spectrum of activities against a wide variety of helminths, protozoa, and enteric bacteria infecting animals and humans. NTZ and other thiazolides are active against a wide range of other intracellular and extracellular protozoan parasites in vitro and in vivo, but their mode of action and respective subcellular target(s) have only recently been investigated. In order to identify potential targets of NTZ and other thiazolides in Giardia lamblia trophozoites, we have developed an affinity chromatography system using the deacetylated derivative of NTZ, tizoxanide (TIZ), as a ligand. Affinity chromatography on TIZ-agarose using cell extracts of G. lamblia trophozoites resulted in the isolation of an approximately 35-kDa polypeptide, which was identified by mass spectrometry as a nitroreductase (NR) homologue (EAA43030.1). NR was overexpressed as a six-histidine-tagged recombinant protein in Escherichia coli, purified, and then characterized using an assay for oxygen-insensitive NRs with dinitrotoluene as a substrate. This demonstrated that the NR was functionally active, and the protein was designated GlNR1. In this assay system, NR activity was severely inhibited by NTZ and other thiazolides, demonstrating that the antigiardial activity of these drugs could be, at least partially, mediated through inhibition of GlNR1.     

Nitazoxanide, a nitrothiazolide antiparasitic drug, is an anti-Helicobacter pylori agent with anti-vacuolating toxin activity.

Nitazoxanide (NTZ), a synthesized drug of the nitrothiazolide class, was initially developed as an antiparasitic compound. This compound has recently been shown to have antibacterial activities against some bacterial pathogens. In the present study, NTZ and its main metabolite tizoxanide (TIZ) were found to have strong minimum inhibitory concentrations (MICs) against both metronidazole (MTZ)-resistant strains and sensitive clinical isolates of Helicobacter pylori. The MIC90 of both NTZ and TIZ against 37 clinical isolates was 8 microg/ml. Vacuolating toxin activity of H. pylori assayed by HeLa cell vacuole formation was inhibited by NTZ at a sub-MIC. In contrast, urease production by H. pylori was not specifically affected by the sub-MIC of NTZ. An acidic pH (pH 5.0) medium reduced the antimicrobial activity of the drug in terms of growth inhibition due to the low growth rate of the bacteria, but killing activity of NTZ against the bacteria was still observed. Thus, it was apparent that both NTZ and TIZ are highly effective against H. pylori, even when the bacteria are resistant to MTZ.     

In vitro efficacies of nitazoxanide and other thiazolides against Neospora caninum tachyzoites reveal antiparasitic activity independent of the nitro group

The thiazolide nitazoxanide [2-acetolyloxy-N-(5-nitro-2-thiazolyl)benzamide] (NTZ) exhibits a broad spectrum of activities against a wide variety of intestinal and tissue-dwelling helminths, protozoa, and enteric bacteria infecting animals and humans. The drug has been postulated to act via reduction of its nitro group by nitroreductases, including pyruvate ferredoxin oxidoreductase. In this study, we investigated the efficacies of nitazoxanide and a number of other thiazolides against Neospora caninum tachyzoites in vitro. We employed real-time-PCR-based monitoring of tachyzoite adhesion, invasion, and intracellular proliferation, as well as electron microscopic visualization of the effects imposed by nitazoxanide. In addition, we investigated several modified versions of this drug. These modifications included on one hand the replacement of the nitro group on the thiazole ring with a bromide, thus removing the most reactive group, and on the other hand the differential positioning of methyl groups on the salicylate ring. We show that the thiazole-associated nitro group is not necessarily required for the action of the drug and that methylation of the salicylate ring can result in complete abrogation of the antiparasitic activity, depending on the positioning of the methyl group. These findings indicate that other mechanisms besides the proposed mode of action involving the pyruvate ferredoxin oxidoreductase enzyme could be responsible for the wide spectrum of antiparasitic activity of NTZ and that modifications in the benzene ring could be important in these alternative mechanisms.     

In vitro activity of nitazoxanide and related compounds against isolates of Giardia intestinalis, Entamoeba histolytica and Trichomonas vaginalis.

The activities of the N-(nitrothiazolyl) salicylamide nitazoxanide and its metabolite tizoxanide were compared with metronidazole in vitro in microplates against six axenic isolates of Giardia intestinalis. Tizoxanide was eight times more active than metronidazole against metronidazole-susceptible isolates and twice as active against a resistant isolate. In 10 axenic isolates of Entamoeba histolytica, while tizoxanide was almost twice as active as metronidazole against more susceptible isolates, it was more than twice as active against less susceptible isolates. Fourteen metronidazole-susceptible isolates of Trichomonas vaginalis were 1.5 times more susceptible to tizoxanide, which was nearly five times as active against resistant isolates. Two highly metronidazole-resistant isolates retained complete susceptibility to tizoxanide, and one moderately resistant isolate showed reduced susceptibility. In all three organisms, nitazoxanide results paralleled those of tizoxanide. Analogues lacking the reducible nitro-group had similar low activities against susceptible G. intestinalis, E. histolytica and T. vaginalis, indicating that nitro-reduction and free radical production was a probable mode of action. Nitazoxanide and its metabolite tizoxanide are more active in vitro than metronidazole against G. intestinalis, E. histolytica and T. vaginalis. Although, like metronidazole, they depend on the presence of a nitro-group for activity, they retain some activity against metronidazole-resistant strains, particularly of T. vaginalis. The results suggest that resistance mechanisms for metronidazole can be bypassed by nitazoxanide and tizoxanide.     

Enzymes associated with reductive activation and action of nitazoxanide,nitrofurans, and metronidazole in Helicobacter pylori

Nitazoxanide (NTZ) is a redox-active nitrothiazolyl-salicylamide prodrug that kills Helicobacter pylori and also many anaerobic bacterial, protozoan, and helminthic species. Here we describe development and use of a spectrophotometric assay, based on nitroreduction of NTZ at 412 nm, to identify H. pylori enzymes responsible for its activation and mode of action. Three enzymes that reduce NTZ were identified: two related NADPH nitroreductases, which also mediate susceptibility to metronidazole (MTZ) (RdxA and FrxA), and pyruvate oxidoreductase (POR). Recombinant His-tagged RdxA, FrxA, and POR, overexpressed in nitroreductase-deficient Escherichia coli, each rapidly reduced NTZ, whereas only FrxA and to a lesser extent POR reduced nitrofuran substrates (furazolidone, nitrofurantoin, and nitrofurazone). POR exhibited no MTZ reductase activity either in extracts of H. pylori or following overexpression in E. coli; RdxA exhibited no nitrofuran reductase activity, and FrxA exhibited no MTZ reductase activity. Analysis of mutation to rifampin resistance (Rif(r)) indicated that NTZ was not mutagenic and that nitrofurans were only weakly mutagenic. Alkaline gel DNA electrophoresis indicated that none of these prodrugs caused DNA breakage. In contrast, MTZ caused DNA damage and was strongly mutagenic. We conclude that POR, an essential enzyme, is responsible for most or all of the bactericidal effects of NTZ against H. pylori. While loss-of-function mutations in rdxA and frxA produce a Mtz(r) phenotype, they do not contribute much to the innate susceptibility of H. pylori to NTZ or nitrofurans.     

A new method for assessing metronidazole susceptibility of Giardia lamblia trophozoites

A quantitative, simple, and rapid assay has been developed to assess Giardia lamblia trophozoite sensitivity to metronidazole [1-(2-hydroxyetyl)-2-methyl-5-nitroimidazole] (MTZ). This new assay utilizes the ability of live (surviving) trophozoites to take up oxygen after have been exposed to MTZ. The effect of MTZ on oxygen uptake was compared with its effect on viability as evaluated by a culture method and morphological assays. Oxygen uptake rates decreased in trophozoites treated with MTZ, and this effect was drug concentration dependent: O(2) uptake rates went from 3.04 microM O(2) min(-1) per 10(6) cells to 0.72 microM O(2) min(-1) per 10(6) cells with increasing drug concentration (0.15 to 0.6 mM) in the preincubation. Concentrations of the drug which inhibited oxygen uptake by 28 to 76% in trophozoites killed from 39 to 82% of trophozoites, as evaluated by the culture method, and altered the morphology of 21 to 86% of the trophozoites. Thus, the trophozoites killed by MTZ are nonmotile cells and do not take up oxygen. A good correlation was found between the inhibitory effects of MTZ, as evaluated by oxygen uptake, and cellular viability. Similar 50% inhibitory concentrations were obtained: 0.33 mM by oxygen uptake, 0. 26 mM by the culture method, and 0.35 mM by morphological criteria. Oxygen uptake appears to be a good indicator of parasite viability. Therefore, this new method can provide a convenient means to assess MTZ susceptibility in G. lamblia and can be applied for screening potential antigiardial agents.     

In vitro and in vivo activities of nitazoxanide against Clostridium difficile

We have used the hamster model of antibiotic-induced Clostridium difficile intestinal disease to evaluate nitazoxanide (NTZ), a nitrothiazole benzamide antimicrobial agent. The following in vitro and in vivo activities of NTZ in the adult hamster were examined and compared to those of metronidazole and vancomycin: (i) MICs and minimum bactericidal concentrations (MBCs) against C. difficile, (ii) toxicity, (iii) ability to prevent C. difficile-associated ileocecitis, and (iv) propensity to induce C. difficile-associated ileocecitis. The MICs and MBCs of NTZ against 15 toxigenic strains of C. difficile were comparable to those of vancomycin or metronidazole. C. difficile-associated ileocecitis was induced with oral clindamycin and toxigenic C. difficile in a group of 60 hamsters. Subgroups of 10 hamsters were given six daily intragastric treatments of NTZ (15, 7.5, and 3.0 mg/100 g of body weight [gbw]), metronidazole (15 mg/100 gbw), vancomycin (5 mg/100 gbw), or saline (1 ml/100 gbw). Animals receiving saline died 3 days post-C. difficile challenge. During the treatment period, NTZ (>/=7.5 mg/100 gbw), like metronidazole and vancomycin, prevented outward manifestations of clindamycin-induced C. difficile intestinal disease. Six of ten hamsters on a scheduled dose of 3.0 mg of NTZ/100 gbw survived for the complete treatment period. Of these surviving animals, all but three died of C. difficile disease by between 3 and 12 days following discontinuation of antibiotic therapy. Another group of hamsters received six similar daily doses of the three antibiotics, followed by an inoculation with toxigenic C. difficile. All of the NTZ-treated animals survived the 15-day postinfection period. Upon necropsy, all hamsters appeared normal: there were no gross signs of toxicity or C. difficile intestinal disease, nor was C. difficile detected in the cultures of the ceca of these animals. By contrast, vancomycin and metronidazole treatment induced fatal C. difficile intestinal disease in 20 and 70% of recipients, respectively.     

Structure-activity relationships of pentamidine analogs against Giardia lamblia and correlation of antigiardial activity with DNA-binding affinity.

1,5-Di(4-amidinophenoxy)pentane (pentamidine) and 38 analogs of pentamidine were screened for in vitro activity against the enteric protozoan Giardia lamblia WB (ATCC 30957). All compounds were active against G. lamblia as measured by a [methyl-3H]thymidine incorporation assay. Antigiardial activity varied widely, with 50% inhibitory concentrations (IC50s) ranging from 0.51 +/- 0.13 microM (mean +/- standard deviation) for the most active compound to over 100.0 microM for the least active compounds. The IC50 of the most potent antigiardial agent, 1,3-di(4-amidino-2-methoxyphenoxy)propane compared favorably with the IC50s of the compounds currently used to treat giardiasis, i.e., furazolidone (1.0 +/- 0.03 microM), metronidazole (2.1 +/- 0.80 microM), quinacrine HCl (0.03 +/- 0.02 microM), and tinidazole (0.78 +/- 0.48 microM). A mode of antigiardial activity for these compounds was suggested by the correlation observed between antigiardial activity and the binding of the compounds to calf thymus DNA and poly(dA).poly(dT).     

Antibacterial activities of nitrothiazole against Campylobacter jejuni and Campylobacter coli.

Niridazole (Ambilhar) and three other newly synthesized nitrothiazole derivatives were highly active against 19 microaerophilic campylobacters (minimum concentration required to inhibit 50% of strains [MIC50], 0.0075 to 0.015 mg/liter). There were, however, considerable differences in the susceptibility among strains tested, and one nitrothiazole derivative was rather inactive (MIC50, 2 mg/liter). Nitroimidazole derivatives, such as metronidazole and tinidazole, were less active (MIC50, 2 and 4 mg/liter, respectively). The nitrofuran derivatives, such as nitrofurazone and nitrofurantoin, were also less active (MIC50, 1 mg/liter). Niridazole and another potent nitrothiazole derivative killed the campylobacters rapidly at low concentrations. In contrast, much higher concentrations of metronidazole were required to achieve bactericidal values.     

Nitazoxanide in treatment of Helicobacter pylori: a clinical and in vitro study

Nitazoxanide (NTZ) is an antibiotic with microbiological characteristics similar to those of metronidazole but without an apparent problem of resistance. The aim of this study was the prospective evaluation of NTZ given as a single agent in the treatment of Helicobacter pylori infection. Twenty culture-positive patients with dyspepsia who had previously failed at least one course of H. pylori eradication therapy were enrolled. Subjects received 1 g of NTZ twice daily for 10 days. The safety and tolerability of the drug were assessed by physical examination, monitoring of adverse events, and clinical laboratory evaluation. Urea breath tests (UBTs) were performed 6 weeks posttreatment. H. pylori was isolated from UBT-positive patients by the string test or endoscopy with biopsy, and the MICs for these isolates were compared to those for isolates obtained pretherapy. The levels of tizoxanide, the active deacylated derivative of NTZ, were measured in blood, saliva, and tissue from two patients during treatment. The UBT results were positive for all 20 patients after completion of NTZ therapy. The MIC results demonstrated that the NTZ susceptibilities of none of the strains isolated from the patients posttherapy had changed significantly. No major adverse reactions were observed, but frequent minor side effects were observed. In conclusion, NTZ did not eradicate H. pylori when it was given as a single agent.     

Effects of metronidazole analogues on Giardia lamblia: experimental infection and cell organization

The chemotherapeutic agents used for the treatment of giardiasis are often associated with adverse side effects and are refractory cases, due to the development of resistant parasites. Therefore the search for new drugs is required. We have previously reported the giardicidal effects of metronidazole (MTZ) and its analogues (MTZ-Ms, MTZ-Br, MTZ-N₃, and MTZ-I) on the trophozoites of Giardia lamblia. Now we evaluated the activity of some giardicidal MTZ analogues in experimental infections in gerbils and its effects on the morphology and ultrastructural organization of Giardia. The giardicidal activity in experimental infections showed ED₅₀ values significantly lower for MTZ-I and MTZ-Br when compared to MTZ. Transmission electron microscopy was employed to approach the mechanism(s) of action of MTZ analogues upon the protozoan. MTZ analogues were more active than MTZ in changing significantly the morphology and ultrastructure of the parasite. The analogues affected parasite cell vesicle trafficking, autophagy, and triggered differentiation into cysts. These results coupled with the excellent giardicidal activity and lower toxicity demonstrate that these nitroimidazole derivates may be important therapeutic alternatives for combating giardiasis. In addition, our results suggest a therapeutic advantage in obtaining synthetic metronidazole analogues for screening of activities against other infectious agents.     

Antiparasitic drug nitazoxanide inhibits the pyruvate oxidoreductases of Helicobacter pylori, selected anaerobic bacteria and parasites, and Campylobacter jejuni

Nitazoxanide (NTZ) exhibits broad-spectrum activity against anaerobic bacteria and parasites and the ulcer-causing pathogen Helicobacter pylori. Here we show that NTZ is a noncompetitive inhibitor (K(i), 2 to 10 microM) of the pyruvate:ferredoxin/flavodoxin oxidoreductases (PFORs) of Trichomonas vaginalis, Entamoeba histolytica, Giardia intestinalis, Clostridium difficile, Clostridium perfringens, H. pylori, and Campylobacter jejuni and is weakly active against the pyruvate dehydrogenase of Escherichia coli. To further mechanistic studies, the PFOR operon of H. pylori was cloned and overexpressed in E. coli, and the multisubunit complex was purified by ion-exchange chromatography. Pyruvate-dependent PFOR activity with NTZ, as measured by a decrease in absorbance at 418 nm (spectral shift from 418 to 351 nm), unlike the reduction of viologen dyes, did not result in the accumulation of products (acetyl coenzyme A and CO(2)) and pyruvate was not consumed in the reaction. NTZ did not displace the thiamine pyrophosphate (TPP) cofactor of PFOR, and the 351-nm absorbing form of NTZ was inactive. Optical scans and (1)H nuclear magnetic resonance analyses determined that the spectral shift (A(418) to A(351)) of NTZ was due to protonation of the anion (NTZ(-)) of the 2-amino group of the thiazole ring which could be generated with the pure compound under acidic solutions (pK(a) = 6.18). We propose that NTZ(-) intercepts PFOR at an early step in the formation of the lactyl-TPP transition intermediate, resulting in the reversal of pyruvate binding prior to decarboxylation and in coordination with proton transfer to NTZ. Thus, NTZ might be the first example of an antimicrobial that targets the "activated cofactor" of an enzymatic reaction rather than its substrate or catalytic sites, a novel mechanism that may escape mutation-based drug resistance.     

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

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 micro M for metronidazole and 0.027 and 0.192 micro M 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.     

Activities of tizoxanide and nitazoxanide compared to those of five other thiazolides and three other agents against anaerobic species

Agar dilution was used, and MICs of metronidazole, tizoxanide, nitazoxanide, denitrotizoxanide, RM 4803, RM 4807, RM 4809, RM 4819, amoxicillin-clavulanate, and clindamycin were measured against 412 anaerobes. Nitazoxanide, tizoxanide, RM 4807, and RM 4809 were active against all groups, except for gram-positive non-spore-forming rods with 50% minimum inhibitory concentrations (when the latter were excluded) of 1 to 2 microg/ml and 90% minimum inhibitory concentrations of 4 to 8 microg/ml, respectively. Metronidazole MICs were usually lower against all groups except clostridia.     

In vitro activities of itraconazole, methiazole, and nitazoxanide versus Echinococcus multilocularis larvae

Albendazole (ABZ) and mebendazole are the only drugs licensed for treatment of human alveolar echinococcosis. In order to augment the armamentarium against this deadly disease, we tested a series of drugs for their efficacy against Echinococcus multilocularis larvae. E. multilocularis larvae grown intraperitoneally in Mongolian gerbils were transferred into tissue culture. Vesicles budded from the tissue blocks and after 6 weeks, drugs were added, and the effect on the vesicles was observed. We tested the following drugs at various concentrations: ABZ, artemether, caspofungin, itraconazole (ITZ), ivermectin, methiazole (MTZ), miltefosine, nitazoxanide (NTZ), rifampin, and trimethoprim-sulfamethoxazole. ABZ, ITZ, MTZ, and NTZ effectively destroyed parasite vesicles in this in vitro culture system. At high NTZ doses of 10 microg/ml, disintegration of all vesicles was observed after 7 days and was significantly more rapid than with ABZ at equal concentrations (21 days). After drug discontinuation, regrowth of vesicles occurred between 7 and 14 days for all four drugs, indicating a parasitostatic effect. Combination treatment with NTZ-ABZ at concentrations between 1 and 10 microg/ml for either 3 weeks, 3 months, or 6 months yielded no vesicle regrowth during 8 months after drug discontinuation. The treated larval tissue was injected intraperitoneally into gerbils, and no regrowth of larval tissue was observed, suggesting a parasitocidal effect after combined treatment. ITZ, MTZ, and NTZ are potent inhibitors of larval growth, although they proved to be parasitostatic only. The combination of NTZ plus ABZ was parasitocidal in vitro. Animal experiments are warranted for studies of dose, toxicity, and drug interactions.     

Drug susceptibility testing of anaerobic protozoa

A simple technique for routine, reproducible global surveillance of the drug susceptibility status of the anaerobic protozoa Trichomonas, Entamoeba, and Giardia is described. Data collected using this technique can be readily compared among different laboratories and with previously reported data. The technique employs a commercially available sachet and bag system to generate a low-oxygen environment and log(2) drug dilutions in microtiter plates, which can be monitored without aerobic exposure, to assay drug-resistant laboratory lines and clinically resistant isolates. MICs (after 2 days) of 3.2 and 25 microM indicated metronidazole-sensitive and highly clinically resistant isolates of T. vaginalis in anaerobic assays, respectively. The aerobic MICs were 25 and >200 microM. MICs (1 day) of 12.5 to 25 microM were found for axenic lines of E. histolytica, and MICs for G. duodenalis (3 days) ranged from 6.3 microM for metronidazole-sensitive isolates to 50 microM for laboratory metronidazole-resistant lines. This technique should encourage more extensive monitoring of drug resistance in these organisms.     

Anti-Toxoplasma gondii Activities and Structure-Activity Relationships of Novel Fluoroquinolones Related to Trovafloxacin

Eleven novel fluoroquinolones closely related to trovafloxacin were evaluated for their in vitro activity against Toxoplasma gondii, and their structure-activity relationships were examined. The 50% inhibitory concentration (IC50) of trovafloxacin against T. gondii was 2.93 microM; the IC50 of the 11 analogs ranged from 0.53 to 14. 09 microM. Six analogs had IC50s lower than that of trovafloxacin. Examination of the structure-activity relationships of the compounds revealed that addition of a -CH3 at C-5 of the 1,8-naphthyridone ring, at C-2 of the azabicyclohexane ring, or on the -NH2 at the 6 position of the azabicyclohexane ring resulted in a four- to sixfold increase in activity. Moreover, replacement of 2,4-difluorophenyl by cyclopropyl at N-1 of the 1,8-naphthyridone ring increased activity twofold, and moving the -NH2 one atom further away from the azabicyclohexane ring decreased activity. There was no difference between the naphthyridone and quinolone analogs. These results indicate that structure-activity studies of compounds related to drugs active against T. gondii may be useful in producing compounds with more potent activities against the parasite.     

Association of metronidazole resistance and natural competence in Helicobacter pylori

To study whether the capability of horizontal DNA transfer is associated with metronidazole resistance in Helicobacter pylori, a total of 81 clinical isolates were tested for MICs of metronidazole (MTZ). The MIC assays were performed by using the E-test and reconfirmed by the agar dilution method. Natural competence assays were performed by transferring a chloramphenicol acetyltransferase cassette and a 23S rRNA gene from a clarithromycin-resistant strain (with an A-to-G mutation at nucleotide 2143) by using natural transformation. Of the 81 isolates, 65 (80.2%) were naturally competent while 16 were not. Among the 65 naturally competent strains, 39 (60%) were highly resistant to MTZ (MICs, >32 microg/ml) while only 2 of 16 (12.5%) noncompetent strains were highly MTZ resistant (P, <0.001). Therefore, there is an association between natural competence and MTZ resistance.     

Entamoeba histolytica alcohol dehydrogenase 2 (EhADH2) as a target for anti-amoebic agents

OBJECTIVES: The current use of metronidazole as an anti-amoebic agent causes significant side-effects. The purpose of this study was to identify alternative compounds with which to treat amoebiasis. METHODS: We tested the effects of cyclopropyl (CPC) and cyclobutyl (CBC) carbinols on the survival of Entamoeba histolytica trophozoites and on the enzymatic activities of E. histolytica alcohol dehydrogenase 2 (EhADH2), a crucial enzyme in the amoebic fermentation pathway. RESULTS: At 72 h, the estimated 50% inhibitory concentrations of CPC and CBC were 38.9 and 11.2 microM, respectively. The EhADH2 alcohol and aldehyde dehydrogenase activities were inhibited by 1.82 microM CPC and 0.89 microM CBC in vitro. CONCLUSIONS: CPC and CBC are expected to be non-toxic to humans at the concentrations required to eliminate E. histolytica trophozoites. Similarities between EhADH2 and the Giardia lamblia AdhE enzyme indicate that CPC and CBC could be effective drugs for treatment of both amoebiasis and giardiasis.