Standardizing the assessment of amoebicidal efficacy of contact lens solutions against Acanthamoeba species

Abstract

Background To date no standardized methods are used in order to assess the amoebicidal efficacy of commercial contact lens solutions for both trophozoites and cysts of Acanthamoeba species. Here we present two methods that are suitable for this purpose: The Spearman-Karber log reduction method and XTT colorimetric assay. Methods Acanthamoeba castellanii (ATCC 50370) and A. polyphaga (ATCC 30461) trophozoites were cultured in peptone-yeast extract-glucose medium. Cysts were developed in Neff’s encystement medium for 1 week. Spearman-Karber and XTT colorimetric assay were used to evaluate trophozoite and cystocidal efficacy of multi-purpose contact lens solutions (MPS). Results With trophozoites, the Spearman-Karber method gave a log reduction estimate of morphological kill between log 0,83 and log 3,61 of the various contact lens solutions, enabling the differentiation between efficacious and less efficacious solutions. With cysts the maximum log reduction of 2,17 was achieved for all 3 MPS solutions at 8 hours. The XTT colorimetric assay showed reduction in trophozoite metabolic rates between 50 and 100% as provided by an optical density signal. All lens fluid solution with a reduction rate > 90% showed residual growth of Acanthamoeba after one week of incubation on nutrient agar covered with Enterobacter aerogenes. Conclusion Both methods give reproducible estimates of amoebicidal efficacy of contact lens solutions, however, XTT colorimetric assay should be followed by an assay for residual growth to test for viable cysts.

In Vitro Effectiveness of Soft Contact Lens Solutions Available on the Dutch Market against Acanthamoeba Species

“…Two common laboratory strains of Acanthamoeba species, Acanthamoeba castellanii (ATCC 50370) and Acanthamoeba polyphaga (ATCC 30461), were selected. Trophozoites were grown in peptone yeast extract glucose (PYG) medium (Merck KGaA, Darmstadt, Germany), in T25 tissue culture flasks (Merck KGaA), at 28 °C [ 16 ]. Cysts were obtained by the inoculation of trophozoites into Neff’s constant pH encystment medium (NEM, Merck KGaA) and incubation for 7 days at 28 °C, without agitation [ 17 ].…”

“…A standard solution of 5 × 10 6 cells/mL was prepared for A. castellanii and A. polyphaga , containing either trophozoites or cysts. The two methods are described in greater detail by de Kroon et al, and they were performed in triplicate for the two different strains [ 16 ].…”