Debarun Dutta

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.

Broad spectrum antimicrobial activity of melimine covalently bound to contact lenses.

PURPOSE To develop a stable antimicrobial contact lens, which is effective against the International Organization for Standardization (ISO) panel microorganisms, Acanthamoeba castellanii and drug resistant strains of Pseudomonas aeruginosa and Staphylococcus aureus. METHODS Melimine was covalently incorporated into etafilcon A lenses. The amount of peptide present on the lens surface was quantified using amino acid analysis. After coating, the heat stability (121°C), lens surface hydrophobicity (by captive bubble), and in vitro cytotoxicity to mouse L929 cells of the lenses were investigated. Antimicrobial activity against the micro-organisms was evaluated by viable plate count and fluorescence microscopy, measuring the proportion of cell death compared with control lenses with no melimine. RESULTS The most effective concentration was determined to be 152 ± 44 μg lens(-1) melimine on the lens surface. After coating, lenses were relatively hydrophilic and were nontoxic to mammalian cells. The activity remained high after autoclaving (e.g., 3.1, 3.9, 1.2, and 1.0 log inhibition against P. aeruginosa, S. aureus, A. castellanii, and Fusarium solani, respectively). Fluorescence microscopy confirmed significantly reduced (P < 0.001) adhesion of viable bacteria to melimine contact lenses. Viable count confirmed that lenses were active against all the bacteria and fungi from the ISO panel, Acanthamoeba and gave at least 2 log inhibition against all the multidrug resistant S. aureus and P. aeruginosa strains. CONCLUSIONS Melimine may offer excellent potential for development as a broad spectrum antimicrobial coating for contact lenses, showing activity against all the bacterial and fungal ISO panel microorganisms, Acanthamoeba, and antibiotic resistant strains of P. aeruginosa and S. aureus.

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 activity of four cationic peptides immobilised to poly-hydroxyethylmethacrylate

Abstract 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.

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.

A laboratory assessment of factors that affect bacterial adhesion to contact lenses.

Adhesion of pathogenic microbes, particularly bacteria, to contact lenses is implicated in contact lens related microbial adverse events. Various in vitro conditions such as type of bacteria, the size of initial inoculum, contact lens material, nutritional content of media, and incubation period can influence bacterial adhesion to contact lenses and the current study investigated the effect of these conditions on bacterial adhesion to contact lenses. There was no significant difference in numbers of bacteria that adhered to hydrogel etafilcon A or silicone hydrogel senofilcon A contact lenses. Pseudomonas aeruginosa adhered in higher numbers compared to Staphylococcus aureus. Within a genera/species, adhesion of different bacterial strains did not differ appreciably. The size of initial inoculum, nutritional content of media, and incubation period played significant roles in bacterial adhesion to lenses. A set of in vitro assay conditions to help standardize adhesion between studies have been recommended.

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.

Antimicrobial activity of immobilized lactoferrin and lactoferricin

Lactoferrin and lactoferricin were immobilized on glass surfaces via two linkers, 4-azidobenzoic acid (ABA) or 4-fluoro-3-nitrophenyl azide (FNA). The resulting surfaces were characterized by X-ray photoelectron spectroscopy (XPS) and contact angle measurements. The antimicrobial activity of the surfaces was determined using Pseudomonas aeruginosa and Staphylococcus aureus strains by fluorescence microscopy. Lactoferrin and lactoferricin immobilization was confirmed by XPS showing significant increases (p < 0.05) in nitrogen on the glass surface. The immobilization of both proteins slightly increased the overall hydrophobicity of the glass. Both lactoferrin and lactoferricin immobilized on glass significantly (p < 0.05) reduced the numbers of viable bacterial cells adherent to the glass. For P. aeruginosa, the immobilized proteins consistently increased the percentage of dead cells compared to the total cells adherent to the glass surfaces (p < 0.03). Lactoferrin and lactoferricin were successfully immobilized on glass surfaces and showed promising antimicrobial activity against pathogenic bacteria.

Activity of a melimine derived peptide Mel4 against Stenotrophomonas, Delftia, Elizabethkingia, Burkholderia and biocompatibility as a contact lens coating

PURPOSE To determine the antimicrobial activity of the melimine derived peptide Mel4 against Delftia, Stenotrophomonas, Elizabethkingia, Burkholderia and to investigate biocompatibility of Mel4 as an antimicrobial coating on contact lenses in animals and humans. METHODS In vitro antimicrobial activity of Mel4 was determined against the four Gram negative bacteria by investigating growth curves for 24h followed by viable counts to determine the minimum inhibitory concentration (MIC). Contact lenses were coated by covalently binding Mel4, characterized by amino acid analysis, and were investigated for changes in lens parameters. Safety of Mel-4 coated lenses were determined in a rabbit model of daily contralateral wear. A prospective, randomised, double-masked, contralateral, 1week daily wear human clinical trial was used to evaluate subjective responses and ocular physiology. RESULTS Mel4 was active against all the bacteria tested (MIC50 ranged from 31-1000μgml-1) and produced an antimicrobial surface on contact lenses. Mel4-coating resulted hydrophilic surface without any significant change in contact lens parameters, and showed no signs of cytotoxicity or ocular irritation during rabbit wear. During human clinical trial, there were no differences between Mel4 coated and uncoated contact lenses in lens performance indicators and ocular signs such as corneal fluorescein staining. Mel4 and control uncoated lenses had no differences in ocular symptoms during lens wear. CONCLUSION Mel4 has achieved antimicrobial activity against variety of Gram negative bacteria that are often resistant to the action of cationic peptides and have been implicated in contact lens related adverse events. Mel4-coated contact lenses were safe to wear.

Comparability and repeatability of pachymetry in keratoconus using four noncontact techniques.

Purpose: To compare and determine the repeatability of central corneal thickness (CCT) measurements using four noncontact pachymetry instruments in eyes with keratoconus. Materials and Methods: The CCT of consecutive patients with keratoconus was measured during a single visit using the swept source optical coherence tomography (SS-OCT, Casia SS-1000°CT, Tomey, Nagoya, Japan), a rotating Scheimpflug camera system (Pentacam, Oculus Optikgerate GmbH, Wetzlar, Germany), scanning slit topographer (Orbscan IIz topography, Baush and Lomb Surgical Inc., San Dimas, CA, USA), and a hand-held spectral domain OCT (HHSD-OCT, Bioptigen Inc., Durham, North Carolina, USA). Test-retest variability, correlation between measurements and interdevice agreement were analyzed. Results: Fifty eyes of 25 participants were analyzed in this study. All measurement methods correlated well with each other (r > 0.9, P < 0.001). Mean ± standard deviation CCT measured by HHSD-OCT, Orbscan IIz, SS-OCT, and Pentacam was 462 ± 41 μm, 458 ± 41 μm, 454 ± 40 μm, and 447 ± 42 μm, respectively. While the HHSD-OCT over-estimated the CCT (P < 0.001), there was a good correlation between the measurements obtained from the other three devices. However, the numerical difference was high and this trend was seen in all the paired comparisons. Conclusions: Though the measurements by different devices correlated well, the numerical agreement may be inadequate for their interchangeable use in clinical practice.