Different epithelial cleavage planes produced by various epikeratomes in epithelial laser in situ keratomileusis.

PURPOSE: To determine the anatomic cleavage planes produced by various epikeratomes in epithelial laser in situ keratomileusis (epi-LASIK). SETTING: Department of Ophthalmology, IIsan Paik Hospital, Goyang, Korea. METHODS: Sixteen eyes (8 patients) were included in this study. Three epikeratomes, the Moria Epi-K, Centurion SES, and Amadeus II, were used to collect 4 epithelial flaps from 2 patients in the epi-LASIK procedure. Four epithelial flaps from 2 patients were also obtained by laser-assisted subepithelial keratectomy (LASEK). Immunohistochemical staining using monoclonal antibodies against integrin beta1, integrin beta4, laminin 5, and collagen type VII was performed. RESULTS: Immunohistochemical staining showed expression of integrin beta1 and integrin beta4 in all epithelial flaps. In epi-LASIK, the expression of laminin 5 and collagen type VII had a linear or dotted pattern that differed based on the epikeratome used. In the epithelial flaps obtained using LASEK, the expression of laminin 5 and collagen type VII had a dotted pattern. CONCLUSIONS: Each epikeratome yielded reproducible but different cleavage planes of corneal epithelium. The results suggest that further study is needed to elucidate the wound-healing process after epi-LASIK because different cleavage planes produced by different epikeratomes may influence the process.

Laboratory investigation of Acanthamoeba lugdunensis from patients with keratitis.

PURPOSE: Species of four Acanthamoeba isolates (KA/E2, KA/E12, KA/E15, and KA/E16) from the cornea of patients with keratitis were identified and their molecular characteristics compared with those of other strains. METHODS: Morphologic features of amebic cysts were evaluated with a microscope with differential interference contrast (DIC) optics. Restriction fragment length polymorphisms (RFLP) of mitochondrial DNA (mtDNA), riboprinting of small subunit ribosomal RNA gene (18S rDNA), and DNA sequences of 18S rDNA were analyzed. mtDNA and PCR-amplified 18S rDNA of the ocular isolates were digested with restriction enzymes, and the restriction patterns were compared with those of reference strains purchased from American Type Culture Collection (ATCC, Manassas, VA). PCR products of 18S rDNA were cloned and subjected to sequencing. The complete sequence of approximately 2300 bp obtained from the isolates and reference strains were compared with each other and those registered in GenBank. RESULTS: Three ocular isolates (KA/E2, KA/E12, and KA/E16) of Acanthamoeba revealed the identical mtDNA RFLPs and riboprint patterns with Acanthamoeba L3a, the type strain of A. lugdunensis. The other isolate (KA/E15) had riboprint patterns very similar to A. lugdunensis L3a but quite different mtDNA RFLP patterns from those of all the other strains. A dendrogram based on the riboprint data showed that three ocular isolates were identified as A. lugdunensis and the other isolate was very closely related to this species. Identification of the isolates as A. lugdunensis was confirmed by 18S rDNA sequence analysis. The sequence differences of the four isolates from A. lugdunensis L3a was 0.1% to 0.4% (3 to 8/2284 bp) and 1.2% to 1.5% from A. castellanii Castellani. CONCLUSIONS: This is the first report of Acanthamoeba keratitis in Korea caused by A. lugdunensis, which was originally isolated from a freshwater pool in France. Riboprinting can be used as a simple and rapid tool for putative identification of unknown Acanthamoeba ocular isolates.

Twenty percent alcohol toxicity on rabbit corneal epithelial cells: electron microscopic study.

PURPOSE: To evaluate 20% ethanol toxicity on the rabbit corneal epithelium, ethanol-treated rabbit corneas were examined with electron microscopy. METHODS: Rabbit corneas (24 eyes) were treated with 20% ethanol for 30 seconds, 1 minute, and 2 minutes by using LASEK (laser-assisted subepithelial keratectomy) instruments and then washed with sterile water. Zero time, 1, 3, 5 days after ethanol treatment, corneas were excised and examined with scanning and transmission electron microscopy (SEM and TEM). RESULTS: Widespread partial or total damage of microvilli, focal breaks of intercellular junction, and cellular edema was observed. The damage was more severe in corneas with longer ethanol treatment. In corneas with ethanol treatment more than 1 minute, slough of superficial corneal epithelium occurred and progressed with time. Two-minute ethanol treatment resulted in complete destruction of microvilli and significant separation of intercellular junction. These pathologic changes persisted 5 days after ethanol treatment. CONCLUSIONS: From these results, increasing exposure time to ethanol more than 1 minute results in significant damage to superficial corneal epithelium and prolongs its normal recovery time.

New surgical strategy for corneal tattooing using a femtosecond laser.

PURPOSE: The purpose of this study was to describe a new method of femtosecond (FS) laser-assisted corneal tattooing and to report the results of this method in a case series. METHODS: The study involved 6 eyes of 6 patients with total or partial corneal opacity. An FS laser was used for lamellar dissection and to make a side cut with a hinge, creating a corneal flap. Laser parameters were adjusted depending on ocular condition and the location of the corneal opacity. After lifting the corneal flap, further lamellar dissection with a diamond blade was performed up to the periphery of the limbal area. Using a cannula, dye was injected into the lamellar stromal bed and the space of the lamellar dissected pocket in the limbus, after which the flap was repositioned. RESULTS: The design and application of the FS laser for dissection and flap creation were successful in all cases. There were no significant complications other than an incomplete cut under band keratopathy in 1 eye. The cosmetic effects were satisfactory in all cases. No patient complained of pain for more than 1 day after surgery. CONCLUSIONS: The advantages of the FS laser-assisted corneal tattooing include a customized design, speed, decreased pain, reduced risk of perforation, and good wound healing. This procedure provides improvements over current corneal tattooing techniques, although it is relatively expensive.

Keratitis by Acanthamoeba triangularis: report of cases and characterization of isolates.

Three Acanthamoeba isolates (KA/E9, KA/E17, and KA/E23) from patients with keratitis were identified as Acanthamoeba triangularis by analysis of their molecular characteristics, a species not previously recognized to be a corneal pathogen. Epidemiologic significance of A. triangularis as a keratopathogen in Korea has been discussed. Morphologic features of Acanthamoeba cysts were examined under a microscope with differential interference contrast (DIC) optics. Mitochondrial DNA (mtDNA) of the ocular isolates KA/E9, KA/E17, and KA/E23 were digested with restriction enzymes, and the restriction patterns were compared with those of reference strains. Complete nuclear 18S and mitochondrial (mt) 16S rDNA sequences were subjected to phylogenetic analysis and species identification. mtDNA RFLP of 3 isolates showed very similar patterns to those of SH621, the type strain of A. triangularis. 16S and 18S rDNA sequence analysis confirmed 3 isolates to be A. triangularis. 18S rDNA sequence differences of the isolates were 1.3% to 1.6% and those of 16S rDNA, 0.4% to 0.9% from A. triangularis SH621. To the best of our knowledge, this is the first report, confirmed by 18S and 16S rDNA sequence analysis, of keratitis caused by A. triangularis of which the type strain was isolated from human feces. Six isolates of A. triangularis had been reported from contaminated contact lens cases in southeastern Korea.

Acanthamoeba keratitis related to cosmetic contact lenses.

We report a rare case of Acanthamoeba keratitis related to cosmetic contact lenses in both eyes. A 17-year-old girl with a history of wearing cosmetic contact lenses presented with keratitis. She purchased cosmetic contact lenses via the Internet, and followed a contact lens care system irregularly, occasionally using tap water. Cell culture was performed on samples collected from a corneal scraping, the contact lenses and the storage cases. The isolated organism was Acanthamoeba. The patient was treated with polyhexamethylene biguanide and chlorhexidine for 3 months, and recovered with normal visual acuity. Poor hygiene and insufficient disinfection may be major risk factors for Acanthameoba keratitis in cosmetic contact lens wearers. The cosmetic contact lens user should receive professional advice before accessing the lenses, and this must be communicated to the public.

Acanthamoeba keratitis related to orthokeratology.

PURPOSE: To report four cases of Acanthamoeba keratitis related to the overnight wearing of orthokeratology lenses. METHODS: Four patients had histories of overnight wearing of orthokeratology lenses when they presented with corneal ulcers. They had used a contact lens care system irregularly with tap water. RESULTS: The organism isolated by corneal scraping was Acanthamoeba. The patients were treated with polyhexamethylene biguanide (PHMB) and chlorhexidine, resulting in a resolution of ocular inflammation. CONCLUSION: The risk of Acanthamoeba keratitis as a potential complication of overnight orthokeratology should be considered, especially in patients with over one-year duration of contact lens wearing. Careful contact lens management is needed and tap-water rinsing should be eliminated from the lens care regimen.

Mitochondrial DNA restriction fragment length polymorphism (RFLP) and 18S small-subunit ribosomal DNA PCR-RFLP analyses of Acanthamoeba isolated from contact lens storage cases of residents in southwestern Korea.

We applied ribosomal DNA PCR-restriction fragment length polymorphism (RFLP) and mitochondrial DNA (mtDNA) RFLP analyses to 43 Acanthamoeba environmental isolates (KA/LH1 to KA/LH43) from contact lens storage cases in southwestern Korea. These isolates were compared to American Type Culture Collection strains and clinical isolates (KA/E1 to KA/E12) from patients with keratitis. Seven riboprint patterns were seen. To identify the species of the isolates, a phylogenetic tree was constructed based on the comparison of riboprint patterns with reference strains. Four types accounted for 39 of the isolates belonging to the A. castellanii complex. The most predominant (48.8%) type was A. castellanii KA/LH2 type, which had identical riboprint and mtDNA RFLP patterns to those of A. castellanii Castellani, KA/E3 and KA/E8. The riboprint and mtDNA RFLP patterns of the KA/LH7 (20.9%) type were identical to those of A. castellanii Ma, a corneal isolate from the United States. The riboprint and mtDNA RFLP patterns of the KA/LH1 (18.6%) type were the same as those of A. lugdunensis L3a, KA/E2, and KA/E12. The prevalent pattern for each type of Acanthamoeba in southwestern Korea was very different from that from southeastern Korea and Seoul, Korea. It is noteworthy that 38 (88.4%) out of 43 isolates from contact lens storage cases of the residents in southwestern Korea revealed mtDNA RFLP and riboprint patterns identical to those found for clinical isolates in our area. This indicates that most isolates from contact lens storage cases in the surveyed area are potential keratopathogens. More attention should be paid to the disinfection of contact lens storage cases to prevent possible amoebic keratitis.