Acanthamoeba spp. aggregate and encyst on contact lens material increasing resistance to disinfection

Abstract

IntroductionAcanthamoeba keratitis is often caused when Acanthamoeba contaminate contact lenses and infect the cornea. Acanthamoeba is pervasive in the environment as a motile, foraging trophozoite or biocide-resistant and persistent cyst. As contact lens contamination is a potential first step in infection, we studied Acanthamoeba’s behavior and interactions on different contact lens materials. We hypothesized that contact lenses may induce aggregation, which is a precursor to encystment, and that aggregated encystment would be more difficult to disinfect than motile trophozoites.MethodsSix clinically and/or scientifically relevant strains of Acanthamoeba (ATCC 30010, ATCC 30461, ATCC 50370, ATCC 50702, ATCC 50703, and ATCC PRA-115) were investigated on seven different common silicone hydrogel contact lenses, and a no-lens control, for aggregation and encystment for 72 h. Cell count and size were used to determine aggregation, and fluorescent staining was used to understand encystment. RNA seq was performed to describe the genome of Acanthamoeba which was individually motile or aggregated on different lens materials. Disinfection efficacy using three common multi-purpose solutions was calculated to describe the potential disinfection resistance of trophozoites, individual cysts, or spheroids.ResultsAcanthamoeba trophozoites of all strains examined demonstrated significantly more aggregation on specific contact lens materials than others, or the no-lens control. Fluorescent staining demonstrated encystment in as little as 4 hours on contact lens materials, which is substantially faster than previously reported in natural or laboratory settings. Gene expression profiles corroborated encystment, with significantly differentially expressed pathways involving actin arrangement and membrane complexes. High disinfection resistance of cysts and spheroids with multi-purpose solutions was observed.DiscussionAggregation/encystment is a protective mechanism which may enable Acanthamoeba to be more disinfection resistant than individual trophozoites. This study demonstrates that some contact lens materials promote Acanthamoeba aggregation and encystment, and Acanthamoeba spheroids obstruct multi-purpose solutions from disinfecting Acanthamoeba.

A detailed dissection of the expression, localization, structure, and diagnostic potential of cyst wall proteins of the eye pathogen Acanthamoeba

“…We tested rAbs versus the model Neff strain of Ac, as well as Acanthamoeba species/strains from the ATCC collected by Monica Crary, who studies their binding to contact lenses (24,27). Segments of 18S RNA genes were amplified from trophozoites, cloned, and sequenced to confirm the genotype of 11 Acanthamoeba species/strains prior to encystation and testing with rAbs (13)(14)(15)(16).…”

“…Cyst walls, which form when trophozoites are starved of nutrients, protect free-living Acanthamoebae from osmotic shock in freshwater or drying in the air [21,22]. The cyst wall also makes Acanthamoebae resistant to disinfectants used to clean surfaces, alcohol-based hand sanitizers, sterilizing agents in contact lens solutions, and/or antibiotics applied to the eye [23][24][25][26][27]. While trophozoites cause damage to corneal epithelial cells, AK is most often diagnosed by microscopic identification of cysts in corneal scrapings [1,2,28].…”

Biological characteristics and pathogenicity of Acanthamoeba

“…To overcome this issue, second generation silicon hydrogel lenses have been developed and trophozoite attachment rates for this generation of lenses were much lower than for the first generation and not statistically different from those of conventional hydrogel lenses ( Beattie et al, 2006 ). Recently, Campolo et al (2022) found that some contact lens materials are more conducive to cyst formation than the natural environment with encystation occurring within as little as 4 h of incubation. They hypothesized that aggregation of cysts directly obstructs multi-purpose solutions from disinfecting Acanthamoeba , which further indicates that contact lens materials may need to be reevaluated in the future as infection incidence increases ( Campolo et al, 2022 ).…”

“…Recently, Campolo et al (2022) found that some contact lens materials are more conducive to cyst formation than the natural environment with encystation occurring within as little as 4 h of incubation. They hypothesized that aggregation of cysts directly obstructs multi-purpose solutions from disinfecting Acanthamoeba , which further indicates that contact lens materials may need to be reevaluated in the future as infection incidence increases ( Campolo et al, 2022 ). The adhesion of trophozoites and cysts to the surface of CLs depends on the structure of both.…”

Complete Recovery of Acanthamoeba Motility among Surviving Organisms after Contact Lens Care Disinfection

“…Overall, there is a lack of research and understanding regarding Acanthamoeba motility and how it relates to contact lens use, or how it is impacted by CLC disinfection. We recently published a novel method regarding Acanthamoeba motility on control surfaces with and without available nutrients [ 8 ], and found that amoebae were able to remain motile for at least 12 h even in the absence of nutrients [ 9 ]. This indicated that amoebae could potentially travel substantial distances on contaminated lenses, or from a lens to a cornea and into the corneal epithelium, within the normal period of time for either wearing a lens or in which the lens is in its case.…”