Differential Antimicrobial Efficacy of Preservative-Free Contact Lens Disinfection Systems against Common Ocular Pathogens.

Microbial keratitis is a devastating disease that can cause eye damage and blindness and can be the result of infections by several common ocular pathogens. Importantly, some of these pathogens, such as Acanthamoeba, are particularly unsusceptible to biocides in common contact lens care solutions. Therefore, the disinfection efficacy of preservative-free (PF) disinfection systems against bacteria, fungi, and Acanthamoeba trophozoites and cysts should be assessed as products with the most potential to be efficacious against resistant organisms. PF disinfection systems were analyzed for antimicrobial efficacy. These were the one-step (hydrogen peroxide-based) Clear Care and Clear Care Plus systems and the two-step (povidone-iodine-based) Cleadew system. Stand-alone challenges using bacteria, fungi, and Acanthamoeba were prepared according to the International Standards Organization method 14729. These same challenges were also conducted in the presence of the following contact lenses: Boston RGP, Acuvue Oasys, Biofinity, Ultra, and 2-week PremiO. All challenges were performed at the manufacturer's recommended disinfection time. All preservative-free disinfection systems demonstrated similarly high rates of antimicrobial efficacy when challenged with bacteria or fungi, with or without lenses. However, both Clear Care and Clear Care Plus demonstrated significantly greater disinfection efficacy against Acanthamoeba trophozoites and cysts, with and without lenses (P < 0.05). Cleadew efficacy was impacted by the addition of contact lenses, whereas Clear Care/Clear Care Plus maintained similar efficacies in the absence or presence of lenses. While both hydrogen peroxide and povidone-iodine are highly effective against bacteria and fungi, hydrogen peroxide maintains significantly greater disinfection capabilities than povidone-iodine against all forms of Acanthamoeba. IMPORTANCE Understanding the most efficacious products will allow clinicians to best communicate to patients and consumers the safest products on the market to reduce adverse events, including microbial keratitis, during contact lens use.

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

Introduction: Acanthamoeba 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. Methods: Six 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. Results: Acanthamoeba 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. Discussion: Aggregation/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.

Biocidal Efficacy of a Hydrogen Peroxide Lens Care Solution Incorporating a Novel Wetting Agent.

PURPOSE: To compare the antimicrobial effects of CLEAR CARE, a 3% hydrogen peroxide (H2O2) solution formulated for simultaneous cleaning, daily protein removal, disinfection, and storage of soft (hydrophilic) hydrogel, silicone hydrogel, and gas-permeable contact lenses, and CLEAR CARE PLUS, consisting of the 3% H2O2 solution plus a novel wetting agent, polyoxyethylene-polyoxybutylene (EOBO-21). METHODS: Three lots each of the 2 solutions were incubated with 5 compendial microorganisms required by the Food and Drug Administration (FDA) 510(k) and International Organization for Standardization (ISO) 14729 stand-alone procedures, 4 clinical isolates of Gram-positive and Gram-negative bacteria, and trophozoites and cysts of 2 Acanthamoeba strains that are associated with microbial keratitis. Microbial loads were evaluated after disinfection and neutralization. RESULTS: Both solutions exceeded the FDA/ISO stand-alone primary criteria against Gram-positive and Gram-negative compendial bacteria, yeast, and mold after only 1.5-hr disinfection/neutralization. At the recommended minimum disinfection time, bacteria were reduced by 4.4 to 5.1 logs, yeast by 4.4 to 4.9 logs, and mold by 2.9 to 3.5 logs with and without organic soil. In addition, both solutions eliminated or effectively reduced populations of clinically relevant ocular bacterial isolates (4.5-5.0 logs), Acanthamoeba trophozoites (3.4-4.2 logs), and cysts (1.5-2.1 logs). CONCLUSION: Both solutions eliminated or reduced populations of FDA/ISO compendial bacteria and fungi as well as clinically relevant microorganisms and Acanthamoeba trophozoites and cysts. The addition of EOBO-21 to the 3% H2O2 lens care solution had no impact on antimicrobial activity.

Phylogenetic analysis and the evolution of the 18S rRNA gene typing system of Acanthamoeba.

Species of Acanthamoeba were first described using morphological characters including cyst structure and cytology of nuclear division. More than 20 nominal species were proposed using these methods. Morphology, especially cyst shape and size, has proven to be plastic and dependent upon culture conditions. The DNA sequence of the nuclear small-subunit (18S) rRNA, the Rns gene, has become the most widely accepted method for rapid diagnosis and classification of Acanthamoeba. The Byers-Fuerst lab first proposed an Rns typing system in 1996. Subsequent refinements, with an increasing DNA database and analysis of diagnostic fragments within the gene, have become widely accepted by the Acanthamoeba research community. The development of the typing system, including its current state of implementation is illustrated by three cases: (i) the division between sequence types T13 and T16; (ii) the diversity within sequence supertype T2/T6, and (iii) verification of a new sequence type, designated T20. Molecular studies make clear the disconnection between phylogenetic relatedness and species names, as applied for the genus Acanthamoeba. Future reconciliation of genetic types with species names must become a priority, but the possible shortcomings of the use of a single gene when reconstructing the evolutionary history of the acanthamoebidae must also be resolved.

Characterization of the RNA silencing suppression activity of the Ebola virus VP35 protein in plants and mammalian cells.

Ebola virus (EBOV) causes a lethal hemorrhagic fever for which there is no approved effective treatment or prevention strategy. EBOV VP35 is a virulence factor that blocks innate antiviral host responses, including the induction of and response to alpha/beta interferon. VP35 is also an RNA silencing suppressor (RSS). By inhibiting microRNA-directed silencing, mammalian virus RSSs have the capacity to alter the cellular environment to benefit replication. A reporter gene containing specific microRNA target sequences was used to demonstrate that prior expression of wild-type VP35 was able to block establishment of microRNA silencing in mammalian cells. In addition, wild-type VP35 C-terminal domain (CTD) protein fusions were shown to bind small interfering RNA (siRNA). Analysis of mutant proteins demonstrated that reporter activity in RSS assays did not correlate with their ability to antagonize double-stranded RNA (dsRNA)-activated protein kinase R (PKR) or bind siRNA. The results suggest that enhanced reporter activity in the presence of VP35 is a composite of nonspecific translational enhancement and silencing suppression. Moreover, most of the specific RSS activity in mammalian cells is RNA binding independent, consistent with VP35's proposed role in sequestering one or more silencing complex proteins. To examine RSS activity in a system without interferon, VP35 was tested in well-characterized plant silencing suppression assays. VP35 was shown to possess potent plant RSS activity, and the activities of mutant proteins correlated strongly, but not exclusively, with RNA binding ability. The results suggest the importance of VP35-protein interactions in blocking silencing in a system (mammalian) that cannot amplify dsRNA.

Isolation, morphologic, serologic and molecular identification of Acanthamoeba T4 genotype from the liver of a Temminck's tragopan (Tragopan temminckii).

Members of the genus Acanthamoeba are usually free-living amoebae that are found in a variety of ecological niches including soil, fresh and brackish water, dust in the air, heating, ventilating, and air conditioning filters, swimming pools and hot tubs. Occasionally they are also known to cause central nervous system infections in humans and animals. We isolated into culture an amoeba from the liver of a Temminck's tragopan (horned pheasant) (Tragopan temminckii) that died of amoebic infection. We identified the infecting amoeba as Acanthamoeba sp. based on culture characteristics, cyst morphology and immunofluorescence assays. Additionally, we identified the amoeba as Acanthamoeba, genotype T4, by sequencing a diagnostic region of the nuclear small subunit ribosomal RNA gene.