Ajay Kumar Vijay

In Vivo Performance of Melimine as an Antimicrobial Coating for Contact Lenses in Models of CLARE and CLPU

PURPOSE One strategy to minimize bacteria-associated adverse responses such as microbial keratitis, contact lens-induced acute red eye (CLARE), and contact lens induced peripheral ulcers (CLPUs) that occur with contact lens wear is the development of an antimicrobial or antiadhesive contact lens. Cationic peptides represent a novel approach for the development of antimicrobial lenses. METHODS A novel cationic peptide, melimine, was covalently incorporated into silicone hydrogel lenses. Confirmation tests to determine the presence of peptide and anti-microbial activity were performed. Cationic lenses were then tested for their ability to prevent CLPU in the Staphylococcus aureus rabbit model and CLARE in the Pseudomonas aeruginosa guinea pig model. RESULTS In the rabbit model of CLPU, melimine-coated lenses resulted in significant reductions in ocular symptom scores and in the extent of corneal infiltration (P < 0.05). Evaluation of the performance of melimine lenses in the CLARE model showed significant improvement in all ocular response parameters measured, including the percentage of eyes with corneal infiltrates, compared with those observed in the eyes fitted with the control lens (P < or = 0.05). CONCLUSIONS Cationic coating of contact lenses with the peptide melimine may represent a novel method of prevention of bacterial growth on contact lenses and consequently result in reduction of the incidence and severity of adverse responses due to Gram-positive and -negative bacteria during lens wear.

Ability of silver-impregnated contact lenses to control microbial growth and colonisation

Purpose To examine the ability of silver nano-particles to prevent the growth of Pseudomonas aeruginosa and Staphylococcus aureus in solution or when adsorbed into contact lenses. To examine the ability of silver nano-particles to prevent the growth of Acanthamoeba castellanii.

Importance of rub and rinse in use of multipurpose contact lens solution.

Purpose. The introduction of contact lens multipurpose disinfection solution (MPDS) that can be used in conjunction with a “no-rub” regimen has simplified lens care requirements. Once adhered to a surface, microorganisms can become less susceptible to disinfection. The aim of the study was to evaluate the effect of various regimen steps on the efficacy of MPDS when used with silicone hydrogel and conventional lenses. Methods. Commercially available MPDSs containing polyquad or polyhexamethylene biguanide were used in conjunction with two types of silicone hydrogel (lotrafilcon B and galyfilcon A) and one type of conventional soft contact lenses (etafilcon A). Challenge microorganisms included Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Serratia marcescens ATCC 13880, Fusarium solani ATCC 36031, Candida albicans ATCC 10231, or Acanthamoeba polyphaga Ros. The effect of regimen steps “rub and rinse,” “rinse-only,” or “no rub and no rinse” on the disinfection efficacy of test MPDSs was examined using the ISO 14729 Regimen Test procedure. Results. Overall, the greatest efficacy of MPDSs was observed when “rub and rinse” was performed before disinfection with each of the microorganisms tested, regardless of lens type. “No rub and no rinse” steps resulted in a greater load of microorganisms remaining on lenses compared with the other regimens (p < 0.05). When “rinse-only” was performed before disinfection, the MPDS containing polyquad performed generally better (p < 0.05) than MPDSs containing polyhexamethylene biguanide against bacteria. Significantly, less microorganisms were recovered from galyfilcon A than from other lenses (p < 0.05) when MPDSs were used with “rinse-only” step. Conclusions. This study has demonstrated that “rub and rinse” is the most effective regimen and should be recommended in conjunction with all multipurpose lens care solutions and all contact lens types, particularly with silicone hydrogel lenses.

Bacterial Adhesion to Unworn and Worn Silicone Hydrogel Lenses

Purpose. The objective of this study was to determine the bacterial adhesion to various silicone hydrogel lens materials and to determine whether lens wear modulated adhesion. Methods. Bacterial adhesion (total and viable cells) of Staphylococcus aureus (31, 38, and ATCC 6538) and Pseudomonas aeruginosa (6294, 6206, and GSU-3) to 10 commercially available different unworn and worn silicone hydrogel lenses was measured. Results of adhesion were correlated to polymer and surface properties of contact lenses. Results. S. aureus adhesion to unworn lenses ranged from 2.8 × 104 to 4.4 × 105 colony forming units per lens. The highest adhesion was to lotrafilcon A lenses, and the lowest adhesion was to asmofilcon A lenses. P. aeruginosa adhesion to unworn lenses ranged from 8.9 × 105 to 3.2 × 106 colony forming units per lens. The highest adhesion was to comfilcon A lenses, and the lowest adhesion was to asmofilcon A and balafilcon A lenses. Lens wear altered bacterial adhesion, but the effect was specific to lens and strain type. Adhesion of bacteria, regardless of genera/species or lens wear, was generally correlated with the hydrophobicity of the lens; the less hydrophobic the lens surface, the greater the adhesion. Conclusions. P. aeruginosa adhered in higher numbers to lenses in comparison with S. aureus strains, regardless of the lens type or lens wear. The effect of lens wear was specific to strain and lens. Hydrophobicity of the silicone hydrogel lens surface influenced the adhesion of bacterial cells.

Guinea pig models of acute keratitis responses.

Purpose: Contact lens acute red eye (CLARE) and infiltrative keratitis (IK) are inflammatory responses of the eye associated with extended wear of soft contact lenses and are associated with bacterial colonization of contact lenses. Development of animal models to study these conditions is of importance in confirming risk factors and developing preventative strategies. Methods: Silicone hydrogel contact lenses contaminated with a human CLARE isolate of Pseudomonas aeruginosa at inocula ranging from 1 × 105 to 6 × 106 colony-forming units (CFUs) for 15 minutes, 1 hour, or 24 hours were worn by guinea pigs for times up to 48 hours in 3 models designed to simulate contamination during wear and contact lens care regimens. Eyes were examined by slit-lamp biomicroscopy and histology to evaluate ocular responses and inflammation. Results: Three different models for the effects of bacterial contamination on the etiology of CLARE/IK were developed and tested in the guinea pig eye. Bacterial contamination of contact lenses was confirmed to be a major risk factor for the development of CLARE/IK and a dose response shown. The pathological features of CLARE/IK in the guinea pig were virtually identical to those observed in human eyes. Conclusion: The models characterized here provide a useful tool for development of interventional and therapeutic strategies for these important ocular inflammatory conditions.

Contact lens storage case hygiene practice and storage case contamination.

Objective: Persistent microbial contamination of contact lens storage cases is common and is associated with microbial keratitis and sterile corneal infiltrates. This study investigated the ability of various currently practiced storage case cleaning techniques in the presence and absence of disinfectants to remove robust microbial biofilms. Methods: Test storage cases were inoculated with 2 mL of 108 colony-forming units per milliliter (CFU/mL) of ocular isolates of Pseudomonas aeruginosa or Staphylococcus aureus and incubated for 48 hr. Cases were subsequently treated in a variety of ways that may represent current practice including a 10-s rinse (hot water or multipurpose solution [MPS, containing PMHB and polyquaternium]), followed by air-drying for 6 hr alone, or air-drying and tissue wiping. The number of survivors was enumerated using standard culture techniques. Results: Challenge biofilms contained 8.4±0.1 log CFU (P. aeruginosa) and 7.1±0.2 log CFU (S. aureus). Rinsing with MPS or hot water and air-drying cases had no significant effect on S. aureus biofilms and resulted in only partial removal of P. aeruginosa biofilms (3.2–6.8 log CFU survivors). Rinsing with MPS, tissue wiping, and air-drying showed the greatest reduction in biofilm (0.9±0.2 log CFU survivors of P. aeruginosa and 3.4±1.2 log CFU of S. aureus). Conclusions: Biofilms formed by the S. aureus isolate were less dense but more resistant to hygiene procedures than those of the P. aeruginosa isolate. Rinsing (with MPS or hot water) followed by 6 hr of air-drying was insufficient to remove these heavy biofilms. Rinsing using the MPS followed by tissue wiping and air-drying was the most effective practice for both strains.

Inflammasomes, the eye and anti-inflammasome therapy

Inflammasomes, key molecular regulators that play an important role in inflammation, consist of a central protein, an adaptor protein ASC (apoptosis speck-like protein) and a caspase-1 protein. Upon activation, caspase-1 induces maturation of cytokines such as interleukin-1β (IL-1β) and interleukin-18 (IL-18). The release of these cytokines can result in inflammation. Inflammasomes are activated by a variety of factors and their activation involves complex signalling leading to resolution of infection, but can also contribute to the pathology of inflammatory, autoimmune, and infectious diseases. The role of NLRP1, NLRP3, NLRC4 and AIM2 inflammasomes in the pathogenesis of ocular diseases such as glaucoma, age related macular degeneration (AMD), diabetic retinopathy, dry eye and infections of the eye has been established over the past decade. In experimental studies and models, inhibition of inflammasomes generally helps to reduce the inflammation associated with these eye diseases, but as yet the role of these inflammasomes in many human eye diseases is unknown. Therefore, a need exists to study and understand various aspects of inflammasomes and their contribution to the pathology of human eye diseases. The goal of this review is to discuss the role of inflammasomes in the pathology of eye diseases, scope for anti-inflammasome therapy, and current research gaps in inflammasome-related eye disease.

Melimine-Coated Antimicrobial Contact Lenses Reduce Microbial Keratitis in an Animal Model.

Purpose To determine the ability of antimicrobial peptide melimine-coated contact lenses to reduce the incidence of microbial keratitis (MK) in a rabbit model of contact lens wear. Methods In vitro antimicrobial activity of melimine-coated contact lenses was determined against Pseudomonas aeruginosa by viable count and a radiolabeled assay. The amount of lipopolysaccharide (LPS) associated with bacteria bound to melimine-coated and control lenses was determined. Ocular swabs from rabbit eyes were collected for assessment of ocular microflora. A rabbit model for MK was developed that used overnight wear of contact lenses colonized by P. aeruginosa in the absence of a corneal scratch. During lens wear, detailed ocular examinations were performed, and the incidence of MK was investigated. Bacteria associated with worn lenses and infected corneas were determined by viable plate count. Results Inhibition in viable and total P. aeruginosa adhesion by melimine-coated contact lenses was 3.1 log10 and 0.4 log10, respectively. After colonization, the amount of LPS on lenses was approximately the same with or without melimine. Gram-positive bacteria were found in all the ocular swabs followed by fungus (42%). Melimine-coated lens wear was protective and significantly (odds ratio 10.12; P = 0.012) reduced the incidence of P. aeruginosa-driven MK in the rabbit model. The antimicrobial lenses were associated with significantly (P < 0.001) lower ocular scores, indicating improved ocular signs compared with controls. Conclusions This study showed that contaminated contact lenses can produce MK without corneal epithelial defect in an animal model. Melimine-coated contact lenses reduced the incidence of MK associated with P. aeruginosa in vivo. Development of MK requires viable bacteria adherent to contact lenses, and bacterial debris adherent at the lens surface did not cause keratitis.

Protamine as a potential amoebicidal agent for contact lens disinfection.

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 &mgr;M) 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 &mgr;M 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 &mgr;M 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.

Effect of a warming device on contact lens case contamination.

Purpose: To examine the effectiveness of heating contact lens cases after disinfection on reducing microbial contamination. Methods: One strain each of Pseudomonas aeruginosa (071) and Staphylococcus aureus (31) were used to set up robust biofilms in polypropylene contact lens cases. The effect of dilutions (from 1:10 to 1:1000) of trypticase soy broth (TSB) in phosphate-buffered saline and incubation time (24 to 48h) on the ability of strains to from biofilms with high levels of bacteria were first examined. Then the effect of increasing the temperature of incubation (from 14°C to 60°C) of biofilms during drying was examined. In the final set of experiments, biofilms of strains were subjected to heating in a warming device set to deliver 60°C for 3 hours, and the effect of this temperature after disinfection with a multipurpose disinfecting solution (MPDS; containing polyquat and Aldox) was examined by culturing the number of viable bacterial cells remaining. Results: A dilution of 1:100 TSB for S. aureus 31 and 1:1000 TSB for P. aeruginosa 071 together with an incubation time of 24 hours gave high numbers of viable cells of these 2 strains adhered to the contact lens cases. Having established the biofilms of bacteria, heating these to 60°C for 3 hours resulted in significant reductions in the number of viable cells that could be cultured (1 log reduction for S. aureus 31, P=0.0003; 3.5 log reduction for P. aeruginosa 071, P=0.002). Exposing the biofilms of cells to a disinfection cycle (6h at ambient temperature) in the presence of the MPDS and air drying at ambient temperature resulted in 2441±1237 colony-forming units/lens well for S. aureus 31 and 7401±4374 colony-forming units/lens well for P. aeruginosa 071. Increasing the drying temperature to 60°C resulted in zero viable cells (i.e., ≥4log reduction) for either bacterial type. Conclusions: Using a warming device for contact lens cases after a disinfection cycle with an MPDS during drying for 3 hours results in substantial kill of biofilms of P. aeruginosa and S. aureus that have been formed in the wells of the cases.