Purpose: To evaluate the amoebicidal efficacy of protamine with polyhexamethylene biguanide (PHMB) and ethylenediamine tetraacetic acid (EDTA).
Methods: The International Organization for Standardization 14729:2001 procedure was modified to test amoebicidal activity. Acanthamoeba cells were inoculated into dilutions of protamine alone (57 to 228 μM) or in combination with PHMB/EDTA and incubated at 25°C for 6 hours. The number of survivors was determined after 7 days of incubation at 32°C on Escherichia coli–seeded agar plates. For encystment, Acanthamoeba trophozoites were incubated in protamine/PHMB/EDTA for 24 hours, and then the number of cysts was counted using a hemocytometer.
Results: Protamine showed significant (p < 0.01) activity against trophozoites of both Acanthamoeba strains, which reached 2 log reductions or more for 228 μM compared with that in phosphate buffered saline. The addition of PHMB to protamine significantly (p = 0.002) improved anti-Acanthamoeba effect (0.8 logs reduction) of Acanthamoeba castellanii only. The addition of EDTA to protamine/PHMB only slightly improved efficacy (0.1 logs). Protamine at 228 μM significantly (p < 0.0001) killed the cysts of either strain by between 0.6 and 0.9 logs. Protamine/PHMB significantly increased killing (p = 0.014) of cysts of A. castellanii only. Protamine/PHMB/EDTA did not show synergy against Acanthamoeba cysts. Protamine or protamine/PHMB with or without EDTA did not cause encystment.
Conclusions: Protamine shows good activity against Acanthamoeba trophozoites and cysts and works more effectively in combination with PHMB against A. castellanii. Protamine may be a promising ingredient in contact lens–disinfecting solutions to control Acanthamoeba growth.