A New Method for Assessing Metronidazole Susceptibility of Giardia lamblia Trophozoites

Abstract

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: O2 uptake rates went from 3.04 μM O2 min−1 per 106 cells to 0.72 μM O2 min−1 per 106 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.

Anti-Giardia activity of phenolic-rich essential oils: effects of Thymbra capitata, Origanum virens, Thymus zygis subsp. sylvestris, and Lippia graveolens on trophozoites growth, viability, adherence, and ultrastructure

“…Growth inhibition assay Susceptibility of G. lamblia growth to essential oils in vitro was determined as previously described (Sousa and Poiares-Da-Silva 1999). Essential oils were diluted in DMSO at 100 mg ml −1 and then in TYI-S-33 medium in order to get a range of concentrations from 0.01 to 0.4 mg ml −1 .…”

Evaluation of Giardia duodenalis viability after metronidazole treatment by flow cytometry

“…Increasing drug concentrations and incubation times resulted in decreased numbers of adherent trophozoites and increased PI+ cells ( Figure ). At the longest incubation time, only 10% of the cysts were able to excyst, as previously described ( Sousa & Poiares-da-Silva 1999 , Cruz et al 2003 , Sandhu et al 2004) . After 18 h, at MTZ concentrations > 5.0 µg/mL, more than 90% of cells were dead and, therefore, lost the capacity to grow; these cells were classified as PI+ ( Figure ).…”

Susceptibility of Giardia lamblia to Hovenia dulcis extracts

“…The compounds of choice are 5-nitroimidazole, metronidazole, 5-nitrofuran, furazolidone and more recently, benzimidazoles and pyrantel pamoate. These drugs are described to act on G. lamblia metabolism, reproduction and/or cytoskeleton (Chavez et al 1992;Katiyar et al 1994;Sousa and Silva 1999;Campanati et al 2001;Campanati and MonteiroLeal 2002;MacDonald et al 2004). Although the drugs listed above are effective, many problems have been described in association to their use.…”