Abstract
Purpose: :
The free-living amoeba Acanthamoeba can transform from the trophozoite to a resistant cyst stage. A standardised assay was developed to study factors affecting this response in trophozoites exposed to a reference encystment medium and commercial multipurpose contact lens care solutions (MPS).
Methods: :
A. castellanii (ATCC 50370) and A. polyphaga (ATCC 30461) were studied. A positive encystment control (PEnC) comprised: 0.5% propylene glycol, 0.14% KCl, 0.55% NaCl, 0.05% EDTA and 0.25 ppm PHMB in phosphate buffer, pH 7.0. Trophozoites were inoculated into Ac#6 or PYG axenic media at 1 x 105 /ml, grown for 24 hr at 28ºC before harvesting and washing with ¼ Ringer’s solution by centrifugation at 500 x g for 2 min. Trophozoites were adjusted to 1 x 107 /ml and 30 µl added to 3 ml of test solution containing 25 µg /ml Calcofluor White (CW) in a 12 well microtitre plate. Plates were incubated at 25ºC and the % trophozoite encystment determined from haemocytometer counts performed under phase contrast and UV fluorescence microscopy at 0, 6 and 24 h. Various MPS were also screened by this method.
Results: :
For the PEnC solution, A. castellanii gave 20-30% encystment at 6 hr, rising to 50-70% by 24 hr. For A. polyphaga, the encystment rates were 40-60% at 6 hr and 70-80% at 24 hr. Greater encystment at 6 hr was obtained with both strains grown in PYG compared to Ac#6 medium. Similar results were obtained when the CW was added after incubation. Omission of antibiotics from the growth media gave approximately 10% increased encystment for both species. Assays conducted at 35ºC resulted in reduced encystment levels of at least 50%. Of 8 MPS solutions studied, 2 induced significant and rapid trophozoite encystment. MPS-1 (PHMB, 0.5% propylene glycol, borate buffer) gave 24% (Ac#6) to 60% (PYG) encystment with A. castellanii and 40-60% with A. polyphaga by 6 hr. MPS-2 (PHMB, 0.65% propylene glycol, borate buffer) gave 50-60% at 6 hr for both species and media. Assays conducted in the presence of >100 µg /ml CW resulted one MPS (PQ1, MAPD, 1% propylene glycol) giving 50-85% encystment within 6 hr.
Conclusions: :
A reliable method for determining the rate of Acanthamoeba encystment in response to MPS exposure is described. CW binds to cellulose of the Acanthamoeba inner cyst wall enabling encystment to be observed and recorded in real-time. Of the MPS shown to induce encystment, all contain propylene glycol. The role of MPS induced encystment in the pathogenesis of Acanthamoeba keratitis is unclear but merits investigation.
Keywords: Acanthamoeba • contact lens • clinical laboratory testing