A new quantitative in vitro microculture method for Giardia duodenalis trophozoites

A reliable, rapid and low-cost method for drug sensitivity determination of Giardia duodenalis trophozoites (WB-strain) was developed in a 96-well plate. Using a standard moculum of 5 x 10(4) trophozoites per well (300 mu l), good growth was obtained after sealing the plate with an airtight adhesive tape and incubation at 37 degrees C for 72 h in modified TYI-S-33 medium. Viable burdens were quantified using the formazan dyes NITT (100 mu g/well) and XTT (20 mu g/well) and the fluorescent substrate resazurin (2.5 mu g/well). Prior removal of the culture medium is required since it causes spontaneous reduction of the substrate. Resazurin proved to be far superior to NITT and XTT with a level of sensitivity of about 3 x 104 trophozoites. Inhibitory concentrations (IC50) of several anti-giardial reference drugs were within the range of published values: metronidazole 2.25 mu M, tinidazole 1.75 mu M, albendazole 0.10 mu M, furazolidone 2.00 mu M and quirracrine 0.32 mu M. The broad-spectrum antibiotics chloramphenicol, rifampicin, penicillin G+streptomycin and gentamycin were devoid of any inhibitory activity and are considered suitable for decontamination during excystation experiments. (c) 2007 Elsevier B.V. All rights reserved.