PURPOSE:

Acanthamoeba trophozoites and cysts are ubiquitous throughout nature and known to cause serious corneal infections. To protect contact lens wearers, disinfection protocols require optimization for testing contact lens disinfecting solutions. E. coli (E.c) may be used as a food source to promote recovery of Acanthamoeba organisms after disinfection. Here, the concentration of E.c was optimized to promote optimal recovery of Acanthamoeba cysts following disinfection protocols.

METHODS:

Acanthamoeba castellanii (A.c, ATCC 50370) or Acanthamoeba polyphaga (A.p, ATCC 30461) trophozoites were cultured and seeded onto Non-Nutrient Agar (NNA) plates at 26-30oC. Organisms were harvested and subject to 30 minutes of disinfection by a non-ophthalmic 3% hydrogen peroxide solution. Controls and test samples were serially diluted in 96-well microtitre plates. Three independent E.c (ATCC 8739) cultures were grown overnight and diluted in ¼ Strength Ringers Solution to 100%, 50.0%, 33.3%, 25.0%, 12.5%, and 10.0%, used for recovery procedures and quantified. 50 ul of diluted E.c was added to each well, incubated for 14 days at 26-30oC, and inspected microscopically for recovery. Acanthamoeba recovery values were determined using the Spearman-Karber method and Log10 Reduction (LR) values were calculated.

RESULTS:

Recovery of A.c and A.p trophozoite inoculum controls or after injury were not affected by the E.c concentrations tested with recovery and LR insignificantly different between 100% to 10% E.c dilutions (p>0.05). Recovery of A.c cyst inoculum controls were not significantly different (p>0.05) within the E.c dilutions tested however recovery of A.c after injury were significantly decreased at low E.c concentrations (p<0.05) leading to a significantly elevated lr calculation at e.c dilutions lower than 33.3 25.0 p><0.05, 12.5 p><0.01, 10 p><0.01). a.p cyst inoculum control recovery was significantly lower at e.c dilutions below 25.0 p><0.05) while recovery of a.p cysts after injury were not significantly different p>0.05) leading to significantly lower LR values at E.c dilutions lower than 25.0% (12.5% p<0.05, 10 p><0.01).>

CONCLUSION:

Optimal recovery of Acanthamoeba cysts in disinfection procedures requires the induction of excystment followed by exponential growth. This data supports the use of E.c at concentrations greater than a 33.3% dilution for efficient recovery of Acanthamoeba cysts.