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Contact lens wear is generally safe and provides excellent vision. However, contact lens wear is often associated with the risk of developing ocular surface infection and inflammation, and in severe cases, the infection can result in loss of vision. Antimicrobial peptide-coated contact lenses have been made to help reduce the incidence of infection and inflammation. This paper reviews the research progress from conception, through the laboratory and preclinical tests to the latest information on clinical testing of an antimicrobial contact lens. We provide insights into the pathways followed and pitfalls that have been encountered. The journey has not always been linear or smooth, but has resulted in some of the first published clinical testing of antimicrobial peptide-coated contact lenses in humans. We hope this may help lead to the development and commercialisation of antimicrobial contact lenses in the future.

The design of biocompatible porous scaffolds that encourage cell adhesion for corneal tissue engineering applications continues to be challenging. In addition to porous hydrogels, nanofibers that can simulate the extracellular matrix structure for cell adhesion would be beneficial. Graphite and nano-hydroxyapatite (nHA) are two bioactive materials that have been used to improve cell adhesion in scaffolds for corneal tissue engineering. In this study, nanofibers were fabricated from hydroxyethyl cellulose and polyvinyl alcohol (PVA) and cross-linked by glutaraldehyde bound to graphite and nano-hydroxyapatite. This scaffold surrounded a transparent hydrogel core from PVA that was cross-linked by freeze-thawing cycles. The chemical and mechanical evaluations demonstrated that nanofibers met the requirements as a scaffold for corneal tissue engineering. The results showed that when the concentration of nHA was approximately 1.66 wt%, the morphology of human epithelial cells did not change, and clot formation occurred around the scaffold during the 1-week in vivo implantation.

Catheter-associated biofilms are responsible for a large fraction of hospital acquired infections. Antimicrobial surface coating on catheters providing prevention at source is extensively studied to reduce bacterial adhesion. Antimicrobial peptides such as melimine and Mel4, covalently linked to surfaces, have shown excellent potential in animal and human studies to suppress infection without toxicity. Covalent binding of the peptides on catheter surfaces improves efficacy but so far has been implemented using multistep wet chemical coupling that will impede widespread adoption. Here we demonstrate plasma immersion ion implantation (PIII) as a single step treatment that covalently couples antimicrobial peptides to polyvinyl chloride (PVC). Strong antimicrobial activity was demonstrated by higher than 3 log kill of

Assessment of bacterial dye release following exposure to antimicrobial peptides (AMPs) provides a detailed understanding regarding their interaction with the inner and outer membrane of bacteria, and the leak of bacterial intracellular materials. This underpins the overall antimicrobial mechanism of these membrane-active peptides. DiSC3(5) is a membrane potential sensitive dye and can characterize the changes in bacterial membrane potential following exposure to AMPs (see Note 1). SYTOX Green is a nucleic acid stain that enters the cell upon loss of membrane integrity after exposure to AMPs and binds to DNA. SYTO9 is another nucleic acid stain, whereas propidium iodide (PI) is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. Both of these stains are widely used to monitor the viability of bacteria following exposure to AMPs. This chapter describes the methods of using these as bacterial dye release experiments for assessment of the antimicrobial mechanism of AMPs.

Assessment of bacterial dye release following exposure to antimicrobial peptides (AMPs) provides a detailed understanding regarding their interaction with the inner and outer membrane of bacteria, and the leak of bacterial intracellular materials. This underpins the overall antimicrobial mechanism of these membrane-active peptides. DiSC3(5) is a membrane potential sensitive dye and can characterize the changes in bacterial membrane potential following exposure to AMPs (see Note 1). SYTOX Green is a nucleic acid stain that enters the cell upon loss of membrane integrity after exposure to AMPs and binds to DNA. SYTO9 is another nucleic acid stain, whereas propidium iodide (PI) is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. Both of these stains are widely used to monitor the viability of bacteria following exposure to AMPs. This chapter describes the methods of using these as bacterial dye release experiments for assessment of the antimicrobial mechanism of AMPs.

Infection of the ocular surface can have devastating consequences if not appropriately treated with antimicrobials at an early stage [...]

Melimine and Mel4 are cationic antimicrobial peptides which can resist biofilm development once bound to biomaterials. The aim of the current study was to determine the mode of action of bound melimine and Mel4 against

Pseudomonas aeruginosa is increasingly resistant to conventional antibiotics, which can be compounded by the formation of biofilms on surfaces conferring additional resistance. P. aeruginosa was grown in sub-inhibitory concentrations of the antimicrobial peptides (AMPs) melimine and Mel4 or ciprofloxacin for 30 consecutive days to induce the development of resistance. Antibiofilm effect of AMPs and ciprofloxacin was evaluated using crystal violet and live/dead staining with confocal microscopy. Effect on the cell membrane of biofilm cells was evaluated using DiSC(3)-5 dye and release of intracellular ATP and DNA/RNA. The minimum inhibitory concentration (MIC) of ciprofloxacin increased 64-fold after 30 passages, but did not increase for melimine or Mel4. Ciprofloxacin could not inhibit biofilm formation of resistant cells at 4&times, MIC, but both AMPs reduced biofilms by &gt, 75% at 1&times, MIC. At 1&times, MIC, only the combination of either AMP with ciprofloxacin was able to significantly disrupt pre-formed biofilms (&ge, 61%, p &lt, 0.001). Only AMPs depolarized the cell membranes of biofilm cells at 1&times, MIC either AMP with ciprofloxacin released a significant amount of ATP (p &lt, 0.04), but did not release DNA/RNA. AMPs do not easily induce resistance in P. aeruginosa and can be used in combination with ciprofloxacin to treat biofilm.

Melimine and Mel4 are cationic antimicrobial peptides which can resist biofilm development once bound to biomaterials. The aim of the current study was to determine the mode of action of bound melimine and Mel4 against S. aureus. The peptides were covalently attached to glass using an azidobenzoic acid linker. The amount of attached peptides was confirmed by XPS and amino acid analysis and their covalent attachment by SDS extraction. The release of autolysins after interaction of S. aureus with immobilized peptides was determined in cell free supernatants. The interaction of immobilized peptides with lipoteichoic acid was confirmed by ELISA. Membrane damage by surface bound peptides was assessed using DiSC(3)-5 (membrane potential sensitive), Syto-9 (membrane permeable) and PI (membrane impermeable) dyes with fluorescence microscopy. Release of ATP and nucleic acids (DNA/RNA) was measured in the surrounding fluid. Attachment of the peptides resulted in increased N% for melimine (5.4 ± 1.8%) and for Mel4 (4.8 ± 1.8%). The concentrations of immobilised amino acids were 0.297 nmole for melimine and 0.358 nmole for Mel4. SDS extraction released < 15% of peptides from the glass. The immobilized peptides bound ≥ 4 times more LTA than control surfaces. More autolysins (8 ± 2%; p = 0.026) were released from Mel4 than melimine or control surfaces. Membrane depolarization occurred at 15 min and was associated with a reduction in bacterial viability ≥ 37% for both peptides (p p p = 0.026) and Mel4 gave 17 ± 12% (p = 0.150) PI stained cells after 4 h. DNA/RNA was released only by melimine coatings (2.1 ± 0.1 times; p = 0.011) compared to process control at 6 h. Both bound peptides resulted in the release of ATP, but only melimine released DNA/RNA while Mel4-coating resulted in the release of autolysins. Since the mode of action of melimine and Mel4 relate to the cell surface, they have potential for the development of infection-resistant implants.

The purpose of this study was to investigate the effect of Mel4 antimicrobial peptide-coated contact lenses (MACL) on the microbiota of the conjunctiva and lenses during three months of extended wear.One hundred and seventy-six participants were recruited into a randomised, contralateral, double masked, biweekly extended wear MACL and uncoated control lens trial. At the one month and 3-month visit, the conjunctival microbiota was sampled using sterile cotton swabs and contact lenses were collected aseptically. Standard microbiological procedures were employed for culture of the swabs and contact lenses and identification of the isolated microorganisms.Gram-positive bacteria (predominantly coagulase-negative staphylococci) were the most frequently isolated microbes from both contact lenses and conjunctiva. There was no difference in the frequency of isolation of most bacteria or fungi from the conjunctival swabs of eyes wearing either MACL or control lenses. The only exception was a higher frequency of eyes harbouring Staphylococcus arlettae when wearing control lenses (5%) versus MACL (1%) (p = 0.002). There was no significant difference in the frequency of microbes isolated from MACL or control contact lenses. There were also no differences between lens types in the frequency of isolation of1 microbial type per sampling occasion for either conjunctiva swabs or contact lenses.MACL wear did not change the conjunctival microbiota during extended wear, and the types of microbes isolated from MACL were similar to those isolated from control lenses.

Significance This study investigated the development of an antimicrobial coating on silicone hydrogel contact lenses that may have the capacity to reduce contact lens-related infection and inflammatory events. Purpose The purpose of this study was to develop an effective antimicrobial coating for silicone hydrogel contact lenses by attachment of Mel4 peptide. Methods Lotrafilcon A, comfilcon A, somofilcon A, senofilcon A, and lotrafilcon B silicone hydrogel contact lenses were plasma coated with acrylic acid followed by Mel4 antimicrobial peptide immobilization by covalent coupling. Peptide immobilization was quantified by x-ray electron spectroscopy. Contact lens diameter, base curve, center thickness, and lens surface wettability were measured by captive-bubble contact-angle technique. Antimicrobial activity of the lenses was determined against Pseudomonas aeruginosa and Staphylococcus aureus by viable plate count and also after soaking with artificial tears solution for 1 day. In vivo safety and biocompatibility were determined in an animal model for 1 week. Results Mel4 peptide-coated silicone hydrogel contact lenses were associated with high antimicrobial inhibition (>2 log), except for lotrafilcon B and senofilcon A. Lotrafilcon B did not exhibit any activity, whereas senofilcon A showed 1.4- and 0.7-log inhibition against P. aeruginosa and S. aureus, respectively. X-ray electron spectroscopy revealed significant increases in the lens surface-bound amide nitrogen in all contact lenses except for lotrafilcon B. All contact lens parameters remained unchanged except for the base curve and center thickness for senofilcon A. Mel4 immobilization was associated with a decrease in contact angle. Mel4-coated contact lens wear was not associated with any signs or symptoms of ocular irritation in a rabbit model study. Reduced antimicrobial activity was observed with all the lenses after soaking with artificial tears solution or rabbit wear. Conclusions Mel4 antimicrobial coating may be an effective option for development of antimicrobial silicone hydrogel contact lenses.

Purpose To determine whether Mel4-coated antimicrobial contact lenses (MACLs) can reduce the incidence of corneal infiltrative events (CIEs) during extended wear. Methods A prospective, randomized, double-masked, single-center, contralateral, extended contact lens wear clinical trial was conducted with 176 subjects. Each participant was randomly assigned to wear a MACL in one eye and an uncoated control contact lens in the contralateral eye or an extended-wear biweekly disposable modality for 3 months. The main outcome measures were the incidence of CIEs per 100 eye-months, identification of the microbial types colonizing the contact lenses or eyes at the time of the CIEs, and their susceptibility to Mel4. Results Nine participants (5.1%) experienced unilateral CIEs; six participants had contact lens acute red eye, and three participants had infiltrative keratitis. The incidence rate for CIEs (0.4 events per 100 participant months; 1.7%) in the Mel4-coated lenses (test) was 69% less than that of the control lenses (1.3 events per 100 participant months; 3.4%; P = 0.29). All Gram-negative bacteria isolated from lenses and lids of participants with CIEs (Citrobacter diversus, Acinetobacter haemolyticus, and Acinetobacter lwoffii) were susceptible to Mel4 peptide; minimum inhibitory concentrations ranged from 15.6 to 62.5 µg/mL. Reduction of adhesion of these bacteria by Mel4-coated lenses ranged from 2.1 to 2.2 log10 colony-forming units/lens. Conclusions MACLs had the capacity to reduce CIEs by at least 50% compared with uncoated control lenses during extended wear over 3 months; however, due to the relatively low rates of CIEs, the reduction was not statistically different compared with control lenses. Translational relevance This study provides evidence that antimicrobial contact lenses have the potential to reduce the incidence of corneal infiltrative events during extended wear.

The failure of many antibiotics in the treatment of chronic infections caused by multidrug-resistant (MDR) bacteria necessitates the development of effective strategies to combat this global healthcare issue. Here, we report an antimicrobial platform based on the synergistic action between commercially available antibiotics and a potent synthetic antimicrobial polymer that consists of three key functionalities: low-fouling oligoethylene glycol, hydrophobic ethylhexyl, and cationic primary amine groups. Checkerboard assays with Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli demonstrated synergy between our synthetic antimicrobial polymer and two antibiotics, doxycycline and colistin. Coadministration of these compounds significantly improved the bacteriostatic efficacy especially against MDR P. aeruginosa strains PA32 and PA37, where the minimal inhibitory concentrations (MICs) of polymer and antibiotics were reduced by at least 4-fold. A synergistic killing activity was observed when the antimicrobial polymer was used in combination with doxycycline, killing >99.999% of planktonic and biofilm P. aeruginosa PAO1 upon a 20 min treatment at a polymer concentration of 128 μg mL -1 (4.6 μM) and doxycycline concentration of 64 μg mL -1 (133.1 μM). In addition, this synergistic combination reduced the rate of resistance development in P. aeruginosa compared to individual compounds and was also capable of reviving susceptibility to treatment in the resistant strains.

We are familiar with the use of various ophthalmic biomaterials such as intraocular lenses and contact lenses. However, all these intraocular, periocular, and orbital biomaterials are subject to microbial colonisation and infections that are associated with increased morbidity and cost of ophthalmic care. Development of novel antimicrobial materials for the prevention of such infections is critical to safeguarding vision. In order to achieve this, several antimicrobial strategies have emerged and these are described in this chapter.

Bacterial colonization and biofilm development on medical devices can lead to infection. Antimicrobial peptide-coated surfaces may prevent such infections. Melimine and Mel4 are chimeric cationic peptides showing broad-spectrum antimicrobial activity once attached to biomaterials and are highly biocompatible in animal models and have been tested in Phase I and II/III human clinical trials. These peptides were covalently attached to glass using an azidobenzoic acid linker. Peptide attachment was confirmed using X-ray photoelectron spectroscopy and amino acid analysis. Mel4 when bound to glass was able to adopt a more ordered structure in the presence of bacterial membrane mimetic lipids. The ability of surface bound peptides to neutralize endotoxin was measured along with their interactions with the bacterial cytoplasmic membrane which were analyzed using DiSC(3)-5 and Sytox green, Syto-9, and PI dyes with fluorescence microscopy. Leakage of ATP and nucleic acids from cells were determined by analyzing the surrounding fluid. Attachment of the peptides resulted in increases in the percentage of nitrogen by 3.0% and 2.4%, and amino acid concentrations to 0.237 nmole and 0.298 nmole per coverslip on melimine and Mel4 coated surfaces, respectively. The immobilized peptides bound lipopolysaccharide and disrupted the cytoplasmic membrane potential of Pseudomonas aeruginosa within 15 min. Membrane depolarization was associated with a reduction in bacterial viability by 82% and 63% for coatings melimine and Mel4, respectively (p < 0.001). Disruption of membrane potential was followed by leakage of ATP from melimine (1.5 ± 0.4 nM) or Mel4 (1.3 ± 0.2 nM) coated surfaces compared to uncoated glass after 2 h (p < 0.001). Sytox green influx started after 3 h incubation with either peptide. Melimine coatings yielded 59% and Mel4 gave 36% PI stained cells after 4 h. Release of the larger molecules (DNA/RNA) commenced after 4 h for melimine (1.8 ± 0.9 times more than control; p = 0.008) and after 6 h with Mel4 (2.1 ± 0.2 times more than control; p < 0.001). The mechanism of action of surface bound melimine and Mel4 was similar to that of the peptides in solution, however, their immobilization resulted in much slower (approximately 30 times) kinetics.

Mel4 is a novel cationic peptide with potent activity against Gram-positive bacteria. The current study examined the anti-staphylococcal mechanism of action of Mel4 and its precursor peptide melimine. The interaction of peptides with lipoteichoic acid (LTA) and with the cytoplasmic membrane using DiSC(3)-5, Sytox green, Syto-9 and PI dyes were studied. Release of ATP and DNA/RNA from cells exposed to the peptides were determined. Bacteriolysis and autolysin-activated cell death were determined by measuring decreases in OD620nm and killing of Micrococcus luteus cells by cell-free media. Both peptides bound to LTA and rapidly dissipated the membrane potential (within 30 seconds) without affecting bacterial viability. Disturbance of the membrane potential was followed by the release of ATP (50% of total cellular ATP) by melimine and by Mel4 (20%) after 2 minutes exposure (pM. luteus lawn than melimine treated samples. These findings suggest that pore formation is unlikely to be involved in Mel4-mediated membrane destabilization for Staphylococcci, since there was no significant Mel4-induced PI staining and DNA/RNA leakage. It is likely that the S. aureus killing mechanism of Mel4 involves the release of autolysins followed by cell death. Whereas, membrane interaction is the primary bactericidal activity of melimine, which includes membrane depolarisation, pore formation, release of cellular contents leading to cell death.This work is original, has not been published and is not being considered for publication elsewhere. Part of this manuscript has been presented as a poster presentation in Gordon Research Conference Italy in 2019. There are no conflicts of interest for any of the authors that could have influenced the results of this work. Prof. Mark Willcox holds the patent the for the melimine peptide.

This study examined the prevalence of free-living Acanthamoeba in domestic tap water in the greater Sydney region, Australia, and determined any seasonal variation in prevalence.Fifty-four participants were included in this study following approval from an institutional human research ethics committee. Each participant self-collected two samples (one in summer and another in winter) from the surface of the drain of the bathroom sink using an instructional kit. The samples were cultured by inoculating onto a non-nutrient agar plate seeded with Escherichia coli and incubation at 32°C for two weeks. The plates were microscopically examined for the presence of free-living amoeba. DNA was isolated from 20 samples and a polymerase chain reaction (PCR) assay was performed for amplification of the partial sequence of the 18S ribosomal RNA gene. The PCR amplified products were sequenced using Sanger sequencing and genotyping was performed based on the variation in nucleotide sequences.A total of 97 samples were collected over the two collection periods, with 28.6 per cent of samples morphologically classified as Acanthamoeba. The summer period yielded 16 of 54 (29.6 per cent) samples classified as Acanthamoeba, while the winter period yielded 12 of 43 (27.9 per cent) samples classified as Acanthamoeba. There was no statistically significant difference (p = 0.85) between the prevalence of free-living Acanthamoeba in summer compared to winter. Phylogenetic analysis showed that 15 of 20 (75 per cent) isolates belonged to genotype T4, the most frequent genotype isolated in Acanthamoeba keratitis.The prevalence of free-living Acanthamoeba characterised morphologically in domestic tap water of the greater Sydney region was higher than expected, especially considering the low incidence of Acanthamoeba keratitis in Australia. However, this study did not find variation between seasons. As the T4 genotype was most common, Sydney-based practitioners must always consider Acanthamoeba as a possible causative organism in cases of microbial keratitis, regardless of the season.

Melimine and Mel4 are chimeric cationic peptides with broad-spectrum antimicrobial activity. They have been shown to be highly biocompatible in animal models and human clinical trials. The current study examined the mechanism of action of these two antimicrobial peptides against P. aeruginosa. The effect of the peptides of endotoxin neutralization, and their interactions with cytoplasmic membranes using DiSC(3)-5 and Sytox green, Syto-9 and PI dyes were analysed. Release of ATP and DNA/RNA were determined using ATP luminescence and increase in OD260 nm. The bacteriolytic ability of the peptides was determined by measuring decreases in OD620 nm. Both the peptides neutralized LPS suggesting their interaction with lipid A. Cytoplasmic membrane was disrupted within 30 seconds, which correlated with reductions in cellular viability. At 2 minutes melimine or Mel4, released 75% and 36% cellular ATP respectively (P P. aeruginosa within 5 minutes, started lysis within 2 hours of exposure.

Purpose: To investigate effects of Rose K2 XL semi-scleral contact lenses (Menicon Co.) on visual acuity and higher-order aberrations in eyes with irregular corneas. Methods: One hundred and twelve eyes of 84 patients fitted with Rose K2 XL lenses were analysed. Participants were in 4 clinical groups: keratoconus, intra-corneal ring segments, radial keratotomy, and penetrating keratoplasty. Corrected distance visual acuity and ocular aberrations were determined before lens wear and 60 min into lens wear. The i-Trace aberrometer was used to determine aberrations at 4.5 mm pupil size.Results: There were 55, 22, 19 and 16 eyes in keratoconus, intra-corneal ring segments, radial keratotomy and penetrating keratoplasty groups, respectively. Before lens wear, eyes had poor corrected distance vision acuity (mean and standard deviation + 0.55 +/- 0.33 logMAR), high negative spherical equivalent refraction ( - 6.4 +/- 3.7 D), high cylindrical errors (4.5 +/- 2.2 D), large higher-order root-mean-squared (HO-RMS) aberration (1.5 +/- 1.3 mu m) and large higher-order aberration components. Cylinder was particular high for the penetrating keratoplasty group (mean 5.9 +/- 2.5 D), root-mean-squared third-order coma was lowest for the radial keratotomy group (0.7 +/- 1.0 mu m), and fourth-order spherical aberration was highly negative for the intra-corneal ring segment group (co-efficient -0.4 +/- 0.7 mu m). With lens wear, the values changed considerably. Corrected distance visual acuity improved by 0.51 +/- 0.31 logMAR, cylinder decreased by 3.6 +/- 2.1 D, HO-RMS aberration reduced by 1.1 +/- 1.2 mu m and higher-order aberration components decreased considerably. Magnitudes of group changes reflected the magnitudes before lens wear.Conclusions: Rose K2 XL semi-scleral contact lenses were effective in improving vision and reducing ocular aberrations for eyes with irregular corneas.

Microbes are known to colonize surfaces and form biofilms. These biofilms are communities of microbes encased in a self-produced matrix that often contains polysaccharides, DNA and proteins. Antimicrobial peptides (AMPs) have been used to control the formation and to eradicate mature biofilms. Naturally occurring or synthetic antimicrobial peptides have been shown to prevent microbial colonization of surfaces, to kill bacteria in biofilms and to disrupt the biofilm structure. This review systemically analyzed published data since 1970 to summarize the possible anti-biofilm mechanisms of AMPs. One hundred and sixty-two published reports were initially selected for this review following searches using the criteria ‘antimicrobial peptide’ OR ‘peptide’ AND ‘mechanism of action’ AND ‘biofilm’ OR ‘antibiofilm’ in the databases PubMed; Scopus; Web of Science; MEDLINE; and Cochrane Library. Studies that investigated anti-biofilm activities without describing the possible mechanisms were removed from the analysis. A total of 17 original reports were included which have articulated the mechanism of antimicrobial action of AMPs against biofilms. The major anti-biofilm mechanisms of antimicrobial peptides are: (1) disruption or degradation of the membrane potential of biofilm embedded cells; (2) interruption of bacterial cell signaling systems; (3) degradation of the polysaccharide and biofilm matrix; (4) inhibition of the alarmone system to avoid the bacterial stringent response; (5) downregulation of genes responsible for biofilm formation and transportation of binding proteins.

The objective of this study was to immobilise and characterise a variety of antimicrobial peptides (AMPs) onto poly-hydroxyethylmethacrylate (pHEMA) surfaces to achieve an antibacterial effect. Four AMPs, viz. LL-37, melimine, lactoferricin and Mel-4 were immobilised on pHEMA by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) which assisted covalent attachment. Increasing concentrations of AMPs were immobilised to determine the effect on the adhesion of Pseudomonas aeruginosa and Staphylococcus aureus. The AMP immobilised pHEMAs were characterised by X-ray photoelectron spectroscopy (XPS) to determine the surface elemental composition and by amino acid analysis to determine the total amount of AMP attached. In vitro cytotoxicity of the immobilised pHEMA samples to mouse L929 cells was investigated. Melimine and Mel-4 when immobilised at the highest concentrations showed 3.1 ± 0.6 log and 1.3 ± 0.2 log inhibition against P. aeruginosa, and 3.9 ± 0.6 log and 2.4 ± 0.5 log inhibition against S. aureus, respectively. Immobilisation of LL-37 resulted in up to 2.6 ± 1.0 log inhibition against only P. aeruginosa, but no activity against S. aureus. LFc attachment showed no antibacterial activity. Upon XPS analysis, immobilised melimine, LL-37, LFc and Mel-4 had 1.57 ± 0.38%, 1.13 ± 1.36%, 0.66 ± 0.47% and 0.73 ± 0.32% amide nitrogen attached to pHEMA compared to 0.12 ± 0.14% in the untreated controls. Amino acid analysis determined that the total amount of AMP attachment to pHEMA was 44.3 ± 7.4 nmol, 3.8 ± 0.2 nmol, 6.5 ± 0.6 nmol and 48.9 ± 2.3 nmol for the same peptides respectively. None of the AMP immobilised pHEMA surfaces showed any toxicity towards mouse L929 cells. The immobilisation of certain AMPs at nanomolar concentration to pHEMA is an effective option to develop a stable antimicrobial surface.

Purpose To determine the repeatability of tear ferning (TF) grading analysed by subjective and objective techniques, and its correlation with lipid layer thickness (LLT), non-invasive keratograph break-up time (NIKBUT), and ocular surface disease index (OSDI) comfort scores with and without ocular lubricants. Methods A prospective, randomised, controlled clinical study was conducted. Eighteen healthy non-contact lens wearers aged between 18 to 45 years attended a total of three visits. They were randomly allocated to receive no drops on two occasions to examine method repeatability (visits A and B) or a 60 μL drop of Refresh® (visit C). At each visit, LLT and NIKBUT were assessed followed by basal tear collection. The TF pattern was established by pipetting one μL volume of tears onto a glass slide that was allowed to air-dry and imaged under a microscope. The resulting tear ferning pattern was determined using subjective grading and a novel computerized objective analysis technique that measured the local contrast of ferning contours. Results Paired t-tests found that all measurements collected between eyes, on different days for visit A and B were not significantly different (p >0.05). Eyes and days were averaged and compared to visit C. Paired t-tests between the averaged baseline visits and visit C for LLT, NIKBUT including subjective and objective TF scores showed no significant difference (p>0.05). No significant correlation was shown between the TF scoring and OSDI, LLT or NITBUT (p>0.05). There was however a high significant correlation and agreement between the subjective and objective analysis techniques for TF grading. Conclusions TF grade, LLT and NITBUT are repeatable. As with other ocular surface assessments, subjective and objective TF gradings did not significantly correlate with the measures of tear film stability or comfort. Lubricants had no effect on TF grade, LLT or NITBUT. The novel objective analysis technique is a promising candidate for a repeatable and unbiased assessment of tear ferning.

Purpose. Covalent immobilization of antimicrobial peptide melimine onto contact lenses can produce broad-spectrum antimicrobial lenses. The purpose of this study was to investigate the performance of melimine-coated contact lenses in an animal model and human clinical trial. Methods. Melimine was covalently attached onto the surface of contact lenses via EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) coupling. A rabbit model of daily contralateral wear of lenses for 22 days was conducted to assess the lens safety. A prospective, randomized, double-masked, one-day human clinical trial was used to evaluate subjective responses and ocular physiology during contralateral wear of melimine-coated (test) and uncoated (control) lenses. Delayed reactions were monitored during follow-up visits after 1 and 4 weeks. Ex vivo retention of antimicrobial activity of worn lenses was assessed by reduction in numbers of viable Pseudomonas aeruginosa and Staphylococcus aureus. Results. Melimine-coated lenses produced no ocular signs or symptoms that would indicate cytotoxicity during the lens wear of rabbits. No histological changes were found in rabbit corneas. During the human trial, no differences were observed in wettability, surface deposition, lens-fitting centration, movement, tightness, and corneal coverage between test and control lenses (p 9 0.05). There were no significant differences in bulbar, limbal, or palpebral redness or conjunctival staining (p 9 0.05). Mean corneal (extent, depth, and type) staining was higher for test lenses compared with that for control lenses (p G 0.05). There was no significant difference in subjective responses for lens comfort, dryness, and awareness (p 9 0.05). No delayed reactions were associated with the test lenses. Worn test lenses retained more than 1.5 log inhibition against both bacterial types. Conclusions. Melimine-coated contact lenses were worn safely by humans. However, they were associated with higher corneal staining. The melimine-coated lenses retained high antibacterial activity after wear. (Optom Vis Sci 2014;91:570Y581)

Background Contact lens wear can result in adverse events including bacterially-driven corneal infection and inflammation. These are the result of various kinds of bacteria adhering to contact lenses and either initiating infection of the cornea or producing inflammation of the cornea and conjunctiva. In order to reduce the incidence of these events antimicrobial contact lenses are being developed. In this study, antimicrobial contact lenses containing nanoparticles of silver or copper, or a combination of the two, were produced and evaluated. Methods Silver and copper nanoparticles were produced in polyvinyl alcohol (PVA) polymers by incorporating salts of these metals and then reducing the salts to nanoparticles with sodium hydroxide. Incorporation of nanoparticles into the PVA was confirmed using transmission electron microscopy, attenuated total reflection (ATR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The nanoparticle-containing polymers were then evaluated for physical characteristics such as tensile strength, water content and coloration. PVA containing polymers were evaluated for cytotoxicity to mammalian cells using a standard assay, and for antimicrobial activity using three different assays that measured their ability to inhibit microbial growth on agar plates, inhibit microbial growth in bacterial suspensions, and to inhibit the viability of adherent bacteria. Results Nanoparticles of between 50 and 75 nm were produced in PVA polymers. The production of nanoparticles was also confirmed by characteristic spectral peaks in ATR and XPS. The addition of silver or copper nanoparticles doubled the strength of PVA polymers but halved their elongation before fracture. Silver-containing PVA was cytotoxic but PVA containing copper alone was not cytotoxic. In the agar diffusion assay and inhibition of microbial growth in suspension only silver-containing lenses produced antibacterial activity, but silver and copper nanoparticle-containing lenses reduced bacterial adhesion to lenses. Conclusion both silver and copper nanoparticle-containing lenses were antibacterial, but this depended on the assay used. PVA containing only copper was not cytotoxic. This indicates the copper nanoparticle-containing lenses might be useful to control bacterial colonisation of lenses, and hence the production of bacterially-drive adverse events during lens wear.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Purpose To compare the central and peripheral pachymetry measurements determined using Orbscan IIz (Bausch & Lomb, Rochester, NY), Visante optical coherence tomography (OCT; Carl Zeiss Meditec, Dublin, CA), and RTVue OCT (Oculus Technologies, Wynwood, WA) with ultrasound pachymetry in eyes with established keratoconus and to evaluate the agreement between them. Design Evaluation of diagnostic technologies. Participants One hundred six eyes of 67 consecutive patients with a clinical diagnosis of keratoconus ranging in age from 12 to 40 years. Methods Central corneal thickness (CCT) was determined by all the 4 techniques. Peripheral corneal thicknesses were determined using Orbscan IIz, Visante OCT, and RTVue at 8 points (superior, inferior, temporal, nasal, superior-temporal, inferior-temporal, superior-nasal, and inferior-nasal) all in the 5.0- to 7.0-mm arcuate zone. Main Outcome Measures Central and peripheral keratoconus thickness. Results Ultrasound pachymetry determined significantly higher CCT values than Orbscan IIz ( P P P = 0.037), with a mean ± standard deviation difference of 14±3 μm, 13±2 μm, and 5±3 μm, respectively. The mean CCT difference was minimal (1±3 μm; P = 0.69) between the Orbscan IIz and Visante. A strong correlation was found ( r >0.80) between all the CCT measurement techniques. Orbscan IIz significantly overestimated the peripheral thickness compared with the rest, and the mean differences ranged between 21 and 60 μm. Mean peripheral thickness differences between RTVue and Visante OCT always remained less than 20 μm. Weak correlations and larger limits of agreement were found between the techniques in thinner and peripheral zones. Conclusions Orbscan IIz, Visante, RTVue, and ultrasound pachymetry show high correlation, although Orbscan IIz and Visante significantly underestimated CCT measurements compared with ultrasound pachymetry in keratoconus. Orbscan IIz significantly overestimated peripheral corneal thickness compared with RTVue and Visante. Financial Disclosure(s) The author(s) have no proprietary or commercial interest in any materials discussed in this article.