Human Tear Protein Analysis Using a Quantitative Microfluidic System: A Pilot Study.

OBJECTIVES: Human tears have the potential to be used as biomarkers to aid in the diagnosis and management of dry eye disease (DED). This prospective, controlled pilot study aimed to investigate the hypothesis that a panel of tear protein profiles can be detected and are repeatable when analyzed using a miniaturized quantitative microfluidic system. METHODS: Ten participants were recruited following institutional ethics committee approval. Participants attended two visits 1 week apart when the following measurements were taken in a sequence: tear meniscus height, noninvasive breakup time, ocular redness, tear collection, and corneal and conjunctival staining. Basal tears (>4 µL) were collected using glass microcapillary tubes. Tears were processed to analyze a panel of proteins (14-230 kDa) following the manufacturer's guidelines using a miniaturized quantitative microfluidic system (Protein 230 LabChip with Agilent 2100 Bioanalyzer). Demographics of the clinical measurements and a comparison of the panel of identified proteins and their repeatability were made. RESULTS: Mean age of the participants was 20.8±1.6 years, nine were females, three fulfilled the TFOS DEWS-II diagnostic criteria for DED. The total protein concentration across participants was 6.72±3.56 mg/mL. Several proteins (lysozyme C, lipocalin 1, IgA light chain, zinc-α2-glycoprotein, albumin, and lactoferrin) were identified at both visits for seven or more participants. There were no significant differences ( P >0.05) in individual protein concentrations between the two visits. A high correlation was found between the two visits for all proteins where correlation coefficient ranged between 0.63 and 0.98 ( P <0.05). CONCLUSION: The protein profiles measured by the quantitative microfluidic system are repeatable, thus validating quantitative microfluidic system as a reliable method for investigating a panel of tear proteins. This method is quick, affordable, requires only 4 µL of tear, and is relatively easy method to perform that can be incorporated in a clinical setting. Further studies in larger clinical setting may be beneficial exploring the usability of this method in various patient groups.

Interaction of the surface bound antimicrobial peptides melimine and Mel4 with Staphylococcus aureus.

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 < 0.001). Disruption of the membrane potential resulted in loss of ATP from melimine (0.9 ± 0.4 nM) or Mel4 (0.6 ± 0.3 nM) coated surfaces compared to control (p < 0.001). Melimine coatings yielded 27 ± 11% (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.

Activity of Antimicrobial Peptides and Ciprofloxacin against Pseudomonas aeruginosa Biofilms.

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× MIC, but both AMPs reduced biofilms by >75% at 1× MIC. At 1× MIC, only the combination of either AMP with ciprofloxacin was able to significantly disrupt pre-formed biofilms (≥61%; p < 0.001). Only AMPs depolarized the cell membranes of biofilm cells at 1× MIC. At 1× MIC either AMP with ciprofloxacin released a significant amount of ATP (p < 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.

Lipid Membrane Interactions of the Cationic Antimicrobial Peptide Chimeras Melimine and Cys-Melimine.

Melimine and its derivatives are synthetic chimeric antimicrobial agents based on protamine and melittin. The binding of solubilized melimine and its derivative, with a cysteine on N-terminus, (cys-melimine) on tethered bilayer lipid membranes (tBLMs) was examined using ac electrical impedance spectroscopy. The addition of melimine and cys-melimine initially increased membrane conduction, which subsequently falls over time. The results were obtained for tBLMs comprising zwitterionic phosphatidylcholine, anionic phosphatidylglycerol, or tBLMs made using purified lipids from Escherichia coli. The effect on conduction is more marked with the cysteine variant than the noncysteine variant. The variation in membrane conduction most probably arises from individual melimines inducing increased ionic permeability, which is then reduced as the melimines aggregate and phase-separate within the membrane. The actions of these antimicrobials are modeled in terms of altering the critical packing parameter (CPP) of the membranes. The variations in the peptide length of cys-melimine were compared with a truncated version of the peptide, cys-mel4. The results suggest that the smaller molecule impacts the membrane by a mechanism that increases the average CPP, reducing membrane conduction. Alternatively, an uncharged alanine-replacement version of melimine still produced an increase in membrane conduction, further supporting the CPP model of geometry-induced toroidal pore alterations. All the data were then compared to their antimicrobial effectiveness for the Gram-positive and Gram-negative strains of bacteria, and their fusogenic properties were examined using dynamic light scattering in 1-oleoyl-2-hydroxy- sn-glycero-3-phosphocholine lipid spheroids. We conclude that a degree of correlation exists between the antimicrobial effectiveness of the peptides studied here and their modulation of membrane conductivity.

Development of Silicone Hydrogel Antimicrobial Contact Lenses with Mel4 Peptide Coating.

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.

Esculentin-1a derived peptides kill Pseudomonas aeruginosa biofilm on soft contact lenses and retain antibacterial activity upon immobilization to the lens surface.

Contact lens (CL) wear is a risk factor for development of microbial keratitis, a vision threatening infection of the eye. Adverse events associated with colonization of lenses, especially by the multi-drug resistant and biofilm forming bacterium Pseudomonas aeruginosa remain a major safety issue. Therefore, novel strategies and compounds to reduce the onset of CL-associated ocular infections are needed. Recently, the activity of the frog skin-derived antimicrobial peptide Esc(1-21) and its diastereomer Esc(1-21)-1c was evaluated against both planktonic and sessile forms of this pathogen. Furthermore, Esc(1-21) was found to significantly reduce the severity of P. aeruginosa keratitis in a mouse model and preserve antipseudomonal activity in the presence of human basal tears. Here, we have analyzed the activity of the peptides on P. aeruginosa biofilm formed on soft CLs. Microbiological assays and scanning electron microscopy analysis indicated that the peptides were able to disrupt the bacterial biofilm, with the diastereomer having the greater efficacy (up to 85% killing vs no killing at 4 µM for some strains). Furthermore, upon covalent immobilization to the CL, the two peptides were found to cause more than four log reduction in the number of bacterial cells within 20 minutes and to reduce bacterial adhesion to the CL surface (77%-97% reduction) in 24 hours. Importantly, peptide immobilization was not toxic to mammalian cells and did not affect the lens characteristics. Overall, our data suggest that both peptides have great potential to be developed as novel pharmaceuticals for prevention and treatment of CL-associated P. aeruginosa keratitis.

Antimicrobial activity of four cationic peptides immobilised to poly-hydroxyethylmethacrylate.

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.

Biocompatibility of antimicrobial melimine lenses: rabbit and human studies.

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 > 0.05). There were no significant differences in bulbar, limbal, or palpebral redness or conjunctival staining (p > 0.05). Mean corneal (extent, depth, and type) staining was higher for test lenses compared with that for control lenses (p < 0.05). There was no significant difference in subjective responses for lens comfort, dryness, and awareness (p > 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.

Antimicrobial contact lenses and lens cases: a review.

Antimicrobial agents are being examined with the aim of developing antimicrobial contact lenses and new forms of antimicrobial lens cases. It is hoped that these developments will result in reduced contact lens-related microbial adverse events. In this review, we assess aspects of various antimicrobial strategies, such as cationic metals and peptides, selenium, quorum sensing inhibitors, and various biocidal and non-cidal agents. We highlight the historical challenges, the current scenario of this field, and recommendations for future antimicrobial strategies.

BCLA CLEAR Presbyopia: Management with contact lenses and spectacles.

This paper seeks to outline the history, market situation, clinical management and product performance related to the correction of presbyopia with both contact lenses and spectacles. The history of the development of various optical forms of presbyopic correction are reviewed, and an overview is presented of the current market status of contact lenses and spectacles. Clinical considerations in the fitting and aftercare of presbyopic contact lens and spectacle lens wearers are presented, with general recommendations for best practice. Current options for contact lens correction of presbyopia include soft simultaneous, rigid translating and rigid simultaneous designs, in addition to monovision. Spectacle options include single vision lenses, bifocal lenses and a range of progressive addition lenses. The comparative performance of both contact lens and spectacle lens options is presented. With a significant proportion of the global population now being presbyopic, this overview is particularly timely and is designed to act as a guide for researchers, industry and eyecare practitioners alike.

Corneal thickness in keratoconus: comparing optical, ultrasound, and optical coherence tomography pachymetry.

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<0.001), Visante (P<0.001), and RTVue (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.

Dry eye clinical practice patterns of UK optometrists.

PURPOSE: In light of the increased roles of optometrists working in primary care in the UK, this research study aimed to gain an insight into perceptions of dry eye disease (DED), knowledge and confidence in diagnosis and management, and satisfaction with currently available treatment options. METHODS: Links to an online survey were distributed to optometrists across the UK via optometry websites newsletters, conferences, and local optical committee data bases, between October 2021 and July 2022. The anonymous questionnaire contained a variety of question types including multiple choice, likert-type scale, and free text questions. RESULTS: The survey was completed by 131 optometrists, with a broad range of experience, who reported examining 33.3 ± 31.0 dry eye patients per month. Forty-eight percent of respondents were involved in the provision of an extended service. Fluorescein tear breakup time, corneal fluorescein staining, and anterior lid assessment were the most used clinical procedures, both for diagnosis and monitoring purposes. Sixty percent of respondents reported that they believed their patients were satisfied/managed with artificial tear alone, with the availability of a preservative free option being the top consideration, particularly with increasing severity. Of the 18.7% of respondents who held Independent prescriber status, 68% felt this had widened their ability to diagnose and treat DED. This was evidenced by an increase in steroid recommendation for moderate and severe disease. CONCLUSIONS: Although dry eye disease was perceived to be an important condition, opinions varied widely regarding knowledge and confidence in diagnosis and management. Involvement in an extended service did not alter patient management. However, an increase in therapeutic management and the employment of a stepwise approach to management has been identified.

The effect of non-ablative thermomechanical skin treatment (Tixel®) on dry eye disease: A prospective two centre open-label trial.

PURPOSE: To determine the effects of a thermo-mechanical action-based peri-orbital fractional skin treatment (Tixel®) on dry eye disease. METHODS: This prospective, controlled, open labelled study was conducted at two study centres: Midland Eye, Solihull, UK, and Vallmedic Vision, Andorra. Participants were screened at the baseline visit (visit-1), received three Tixel® treatments at 2-weeks intervals including further assessment (visits 2, 3 and 4). Participants were followed up for three months post-treatment (visit 5). Vision, intraocular pressure (IOP), dry eye symptomatology were assessed, including the Ocular Surface Disease Index (OSDI) questionnaire, non-invasive tear break-up time (NIBUT) and tear osmolarity as well as detailed ophthalmic assessments. RESULTS: Seventy-four participants (41 in Birmingham and 33 in Andorra) with periorbital wrinkles and moderate to severe dry eye disease (DED) were enrolled. The mean age was 59.3 ± 13.3 years and 57 were females. No adverse events, no change in vision (p = 0.310) or IOP (p = 0.419) were observed. Tixel treatment was associated with clinically and statistically significant improvement in the DED symptoms, which was supported by a reduction of 21.40 ± 15.08 (P < 0.001) of the OSDI index. Non-invasive tear break-up time improved by 2.10 ± 0.91 s (p < 0.001) in the Birmingham cohort and 6.60 ± 2.13 s (p < 0.001) in the Andorra cohort. Tear osmolarity reduced from 299.8 ± 13.3 mOsm/L to 298.8 ± 15.6 mOsm/L following the Tixel treatment (p = 0.271). CONCLUSIONS: Thermo-mechanical action-based peri-orbital fractional skin treatment Tixel® could be an attractive, safe and effective treatment for DED. This treatment is associated with high clinical and statistically significant improvement in DED signs and symptoms with no adverse events.

Artificial Tears: A Systematic Review.

Artificial tears are the mainstay of dry eye disease management, but also have a role in corneal abrasion and wound healing, pain and inflammation management, conjunctivitis, keratitis, contact lens rewetting and removal, and foreign body removal. A systematic review of randomized controlled trials (PROSPERO registration CRD42022369619) comparing the efficacy of artificial tears in patients with dry eye to inform prescribing choices using Web of Science, PubMed and Medline databases identified 64 relevant articles. There is good evidence that artificial tears improve symptoms of dry eye disease within a month of regular use, applied about four times a day, but signs generally take several months to improve. Not all patients with dry eye disease benefit from artificial tears, so if there is no benefit over a month, alternative management should be considered. Combination formulations are more effective than single active ingredient artificial tears. Artificial tears containing polyethylene glycol are more effective than those containing carboxymethylcellulose/carmellose sodium and hydroxypropyl methylcellulose. Those classified as having evaporative dry eye disease, benefit from artificial tears with liposomes, especially of higher concentration. The data available is limited by the definition of dry eye disease applied in published studies being variable, as well as the disease severity examined and compliance with artificial tears being rarely quantified.

Effect of Deposition and Protease Digestion on the Ex Vivo Activity of Antimicrobial Peptide-Coated Contact Lenses.

A clinical study of antimicrobial contact lenses containing the cationic peptide Mel4 was conducted. The few adverse events that occurred with this lens occurred on or after 13 nights of wear. The current study examined whether the Mel4 contact lenses lost activity during wear and the mechanism of this loss. Participants wore contact lenses for up to 13 nights. Lenses were tested for their ability to reduce the adhesion of Pseudomonas aeruginosa and Staphylococcus aureus. The amount of protein and lipid extracted from lenses was measured. The ability of trypsin to affect the antimicrobial activity of Mel4-coated contact lenses was measured. Mel4-coated contact lenses lost their antimicrobial activity at six nights of wear for both bacteria. The amount of lipids (13 ± 11 vs. 21 ± 14 µg/lens at 13 nights wear) and proteins (8 ± 4 vs. 10 ± 3 mg/lens at 13 nights of wear) extracted from lenses was not different between Mel4-coated and uncoated lenses, and was not different after three nights when antimicrobial activity was maintained and thirteen nights when they had lost activity (lipid: 25 ± 17 vs. 13 ± 11, p = 0.2; protein: 8 ± 1 vs. 8 ± 4 mg/lens, p = 0.4). Trypsin digestion eliminated the antimicrobial activity of Mel4-coated lenses. In summary, Mel4-coated contact lenses lost antibacterial activity at six nights of wear, and the most likely reason was proteolytic digestion of the peptide. Future studies will design and test proteolytically stable peptide mimics as coatings for contact lenses.

Current approaches to soft contact lens handling training - Global perspectives.

PURPOSE: All neophyte contact lens wearers require training on how to handle contact lenses. Currently, almost no published information exists describing the most common approaches used by those involved in such training in soft contact lens wearers. This study aimed to gather information on the approaches taken by those conducting this training worldwide. METHODS: An online survey was created in English and translated to Spanish and distributed internationally via social media, conference attendees, and professional contacts. The anonymous survey included information on workplace setting of respondents, information about the typical approaches used for application and removal of soft contact lenses, length of the appointment, and success rate with their approach. Survey responses were received between May 2021 and April 2022. RESULTS: A total of 511 individuals completed the survey and responses were received from 31 countries with 48.7% from the UK. The most common approach taught for application was to have the patient hold the upper eyelashes (84.7%) and to hold the lower eyelid with the same hand as the lens (89.4%). Lenses were applied directly to the cornea by 57.7% of the respondents. The most common approach taught for lens removal was to drag the lens inferiorly from the cornea prior to removal (49.3%). Most respondents did not use videos to aid the teaching appointment (62.0%); however, they felt that their approach was successful in most cases (90). Application and removal training sessions lasted a median of 30 min and contact lenses were typically dispensed after the instructor witnessing successful application and removal three times. CONCLUSION: Various methods are adopted globally for training of application and removal of soft contact lenses, with many advising a patient-specific approach is required for success. The results of this survey provide novel insights into soft contact lens handling training in clinical practice.

Novel Peptides with Dual Properties for Treating Pseudomonas aeruginosa Keratitis: Antibacterial and Corneal Wound Healing.

The corneal epithelium is a layer in the anterior part of eye that contributes to light refraction onto the retina and to the ocular immune defense. Although an intact corneal epithelium is an excellent barrier against microbial pathogens and injuries, corneal abrasions can lead to devastating eye infections. Among them, Pseudomonas aeruginosa-associated keratitis often results in severe deterioration of the corneal tissue and even blindness. Hence, the discovery of new drugs able not only to eradicate ocular infections, which are often resistant to antibiotics, but also to elicit corneal wound repair is highly demanded. Recently, we demonstrated the potent antipseudomonal activity of two peptides, Esc(1-21) and its diastereomer Esc(1-21)-1c. In this study, by means of a mouse model of P. aeruginosa keratitis and an in vivo corneal debridement wound, we discovered the efficacy of these peptides, particularly Esc(1-21)-1c, to cure keratitis and to promote corneal wound healing. This latter property was also supported by in vitro cell scratch and ELISA assays. Overall, the current study highlights Esc peptides as novel ophthalmic agents for treating corneal infection and injury, being able to display a dual function, antimicrobial and wound healing, rarely identified in a single peptide at the same micromolar concentration range.

Factors influencing bacterial adhesion to contact lenses.

The process of any contact lens related keratitis generally starts with the adhesion of opportunistic pathogens to contact lens surface. This article focuses on identifying the factors which have been reported to affect bacterial adhesion to contact lenses. Adhesion to lenses differs between various genera/species/strains of bacteria. Pseudomonas aeruginosa, which is the predominant causative organism, adheres in the highest numbers to both hydrogel and silicone hydrogel lenses in vitro. The adhesion of this strain reaches maximum numbers within 1h in most in vitro studies and a biofilm has generally formed within 24 h of cells adhering to the lens surface. Physical and chemical properties of contact lens material affect bacterial adhesion. The water content of hydroxyethylmethacrylate (HEMA)-based lenses and their iconicity affect the ability of bacteria to adhere. The higher hydrophobicity of silicone hydrogel lenses compared to HEMA-based lenses has been implicated in the higher numbers of bacteria that can adhere to their surfaces. Lens wear has different effects on bacterial adhesion, partly due to differences between wearers, responses of bacterial strains and the ability of certain tear film proteins when bound to a lens surface to kill certain types of bacteria.

Bacterial Dye Release Measures in Response to Antimicrobial Peptides.

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.

Ocular Surface Infection and Antimicrobials.

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