Development of a practical complete-kill assay to evaluate anti-Acanthamoeba drugs.

IMPORTANCE: Acanthamoeba keratitis is a debilitating eye disease that requires effective topical drug therapy. Currently, there is no standard in vitro test to evaluate anti-Acanthamoeba drugs. OBJECTIVE: To develop a practical in vitro complete-kill assay to assess anti-Acanthamoeba drugs. DESIGN AND SETTING: Isolates of Acanthamoeba strains (n = 15) evaluated in a clinical laboratory. An in vitro laboratory assay was created to determine whether polyhexamethylene biguanide, 0.02%, chlorhexidine digluconate, 0.02%, hexamidine diisethioonate, 0.1%, and voriconazole, 1.0%, were effective in completely killing 15 different isolates of Acanthamoeba at time points of 24, 48, and 72 hours in comparison with a saline control. Each 0.5-mL volume of drug was inoculated with 0.1 mL of Acanthamoeba cysts (range, 1-3 × 10(6)/mL) (determined with a hemacytometer) and allowed to incubate at 30°C. At the time points listed, 0.05 mL from each treatment group was inoculated onto nonnutrient agar overlaid with Enterobacter aerogenes. The plates were microscopically examined for growth 1 and 2 weeks after inoculation. At 2 weeks, all plates were subcultured onto a fresh medium. At another 7 days, the growth in subculture at each time point was graded "1" for growth and "0" for no growth. MAIN OUTCOMES AND MEASURES: The cumulative grades of 3 time points (range, 0-3) for each drug and isolate were nonparametrically compared to determine differences in growth between the drugs. The "kill" incidence rates over the 3 time points were also compared. RESULTS: In vitro testing determined that antiacanthamoebal efficacy (determined by the median growth grade and the kill incidence rate) was more prominent for hexamidine diisethioonate (median growth grade, 0.0; kill incidence rate, 93% [14 of 15 isolates]) and polyhexamethylene biguanide (median growth grade, 0.0; kill incidence rate, 80% [12 of 15 isolates]) than for chlorhexidine digluconate (median growth grade, 1.0; kill incidence rate, 40% [6 of 15 isolates]), voriconazole (median growth grade, 2.0; kill incidence rate, 13% [2 of 15 isolates]), and saline (median growth grade, 3.0; kill incidence rate, 0% [0 of 15 isolates]). CONCLUSIONS AND RELEVANCE: The complete-kill assay appears to provide separation in the effectiveness of different antiamoebic drug solutions. This assay may be helpful for guiding topical Acanthamoeba therapy and providing a practical method to evaluate and screen new anti-infectives in the treatment of Acanthamoeba keratitis.

Accelerated 20-year sunlight exposure simulation of a photochromic foldable intraocular lens in a rabbit model.

PURPOSE: To assess the long-term biocompatibility and photochromic stability of a new photochromic hydrophobic acrylic intraocular lens (IOL) under extended ultraviolet (UV) light exposure. SETTING: John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, USA. DESIGN: Experimental study. METHODS: A Matrix Aurium photochromic IOL was implanted in right eyes and a Matrix Acrylic IOL without photochromic properties (n = 6) or a single-piece AcrySof Natural SN60AT IOL (n = 5) in left eyes of 11 New Zealand rabbits. The rabbits were exposed to a UV light source of 5 mW/cm(2) for 3 hours during every 8-hour period, equivalent to 9 hours a day, and followed for up to 12 months. The photochromic changes were evaluated during slitlamp examination by shining a penlight UV source in the right eye. After the rabbits were humanely killed and the eyes enucleated, study and control IOLs were explanted and evaluated in vitro on UV exposure and studied histopathologically. RESULTS: The photochromic IOL was as biocompatible as the control IOLs after 12 months under conditions simulating at least 20 years of UV exposure. In vitro evaluation confirmed the retained optical properties, with photochromic changes observed within 7 seconds of UV exposure. The rabbit eyes had clinical and histopathological changes expected in this model with a 12-month follow-up. CONCLUSIONS: The new photochromic IOL turned yellow only on exposure to UV light. The photochromic changes were reversible, reproducible, and stable over time. The IOL was biocompatible with up to 12 months of accelerated UV exposure simulation.

Scanning electron microscopy visualization of methicillin-resistant Staphylococcus aureus after contact with gatifloxacin with and without preservative.

PURPOSE: To test a visual model by looking at the differences in effect of Zymar((R)) (gatifloxacin plus benzalkonium chloride [BAK]) when compared to gatifloxacin and a normal saline (NS) control upon a methicillin and gatifloxacin-resistant Staphylococcus aureus (MRSA) species. METHODS: An ocular isolate of gatifloxacin-resistant (minimal inhibitory concentration >2 to 4 microg/mL) MRSA was grown to confluency. Chambered slides were prepared with bacterial culture smears, and then incubated with either gatifloxacin at the concentrations of 1 and 10 microg/mL, Zymar containing equivalent concentrations of gatifloxacin, or NS. Bacterial cultures were fixed after 10, 30, and 60 min. Fixed slides were coated in gold sputter for examination. Bacteria were visually evaluated with scanning electron microscopy (SEM) at 50,000x. Blinded review of SEM images compared structural changes and mitotic activity across samples. RESULTS: MRSA exposed to 10 microg Zymar for 60 min showed significantly greater pleomorphism and cell wall surface changes when compared to gatifloxacin (P < 0.0001) and NS (P = 0.001), and significantly less mitotic activity than NS (P = 0.002). CONCLUSION: Using SEM, the topical formulation of gatifloxacin 0.3% (Zymar), which contains BAK, had greater antibacterial activity than did gatifloxacin alone in gatifloxacin and methicillin-resistant S. aureus, thereby illustrating potential advantages of the preservative in the commercial formulation. We further show that these effects can be visualized and quantified.

Comparison of the corneal endothelial protective effects of Healon-D and Viscoat.

BACKGROUND: The use of dispersive ophthalmic viscosurgical devices (OVDs) has been shown to provide significant protection against air bubble damage to the corneal endothelium when compared with cohesive OVDs. We compared the corneal endothelial protective effects of a new dispersive OVD, Healon-D, with Viscoat. METHODS: Healon-D and Viscoat were used in a randomized and masked fashion in the anterior chamber of 40 rabbit eyes during a procedure where ultrasound at 70% continuous energy was delivered for 2 min. Two millilitres of air bubbles were injected into the anterior chamber during the first minute of the procedure on each eye. Corneas were then stained with trypan blue and alizarin red and evaluated via light microscopy for endothelial injury. Both denuding of the endothelial layer, as well as damage to endothelial cells were quantified by using the Evaluation of Posterior Capsule Opacification digital imaging system. RESULTS: The denuded area for eyes treated with Healon-D and Viscoat were not significantly different (medians of 0.004167and 0.003333, respectively, P = 0.8908). There was no significant difference in the area of endothelial cell damaged (medians of 0.02183 and 0.01433, respectively, P = 0.4565). When the denuded and damaged areas were calculated together, there was also no difference in the total injured area (medians of 0.05817 and 0.05821, respectively, P = 0.5740). CONCLUSION: The new dispersive OVD Healon-D is equally as effective as Viscoat in protecting the corneal endothelial layer from denuding and damage from air bubbles during anterior segment surgery.

Intraoperative floppy iris syndrome.

PURPOSE OF REVIEW: To describe the condition known as intraoperative floppy iris syndrome in association with systemic alpha-1 blocker use in patients undergoing cataract surgery and techniques to prevent related complications. RECENT FINDINGS: In recent years, an unexpected complication of progressive intraoperative miosis, iris billowing, and prolapse was noted during routine phacoemulsification in patients with current or previous use of alpha-1 adrenergic receptor antagonists. The syndrome is particularly prevalent among patients treated with tamsulosin, an alpha-1A blocker prescribed for the treatment of benign prostatic hyperplasia. This condition is also seen, in smaller numbers, in patients treated with nonspecific alpha-1 blockers. Intraoperative floppy iris syndrome was noted to occur even when patients discontinued alpha-1 blocker medication for years prior to cataract surgery. Several surgical techniques have been successfully employed to circumvent adverse outcomes in patients with known alpha-1 blocker use. However, the risk remains in patients who do not disclose current or prior use of these medications. SUMMARY: There are several clinical concerns, such as most effective methods of managing intraoperative floppy iris syndrome, increasing awareness among physicians and patients about potential risk associated with this class of drug, and importance of disclosure of use prior to cataract surgery.