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

PURPOSE: To report a rare case of Acanthamoeba keratitis in both eyes related to cosmetic contact lenses. METHODS: A 17-year-old girl with a history of wearing cosmetic contact lenses presented with keratitis. She purchased cosmetic contact lenses on the internet, and used contact lens care system irregularly with tap water. RESULTS: After analysis of the corneal scraping, the contact lenses and the storage solution, the organism Acanthamoeba was identified. The patient was treated with polyhexamethylene biguanide (PHMB) and chlorhexidine for 3 months, and recovered with normal visual acuity. CONCLUSIONS: 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 using these lenses, and this precaution must be communicated to the public.

Acanthamoeba Keratitis Related to Orthokeratology Contact Lens

PURPOSE: To report 4 cases of Acanthamoeba keratitis related to orthokeratology lens overnight wear. METHODS: Four patients had histories of overnight orthkeratology lens wear of 10 months to 3 years when they presented with corneal ulcers. RESULTS: The organism isolated by corneal scraping was Aanthamoeba. 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 orthkeratology should be considered.

Evaluation of Donor Corneal Endothelium after Keratoplasty

PURPOSE: To investigate the difference in donor corneal cell changes after penetrating keratoplasty in various corneal diseases. METHODS: Subjects included 36 eyes from 35 people with at least 6 months of follow-up who had undergone penetrating keratoplasty between August 2000 and December 2002. The patients were classified into three groups based on the state of the corneal endothelium. Changes in cell density, polymorphisms, and polymegathism of the donor cornea were compared between groups. Results were analyzed by ANOVA. RESULTS: The overall corneal endothelial cell density after grafting was lower, but the differences in endothelial cell states between the recipient cases were not statistically significant. The change in corneal endothelial cell density showed a significantly higher difference (p=0.0013) when patients had either undergone a rejection episode during recovery or recurred herpetic uveitis. CONCLUSIONS: The preoperative state of the corneal endothelium may affect the survival of donor corneal endothelium after grafting. However, rejection of the transplant contributes more significantly to the survival of the donor corneal endothelium than other factors. We suggest close observation and keen therapy with respect to rejection after grafting.

Evaluation of Donor Corneal Endothelium after Keratoplasty

PURPOSE: To investigate the difference in donor corneal cell changes after penetrating keratoplasty in various corneal diseases. METHODS: Subjects included 36 eyes from 35 people with at least 6 months of follow-up who had undergone penetrating keratoplasty between August 2000 and December 2002. The patients were classified into three groups based on the state of the corneal endothelium. Changes in cell density, polymorphisms, and polymegathism of the donor cornea were compared between groups. Results were analyzed by ANOVA. RESULTS: The overall corneal endothelial cell density after grafting was lower, but the differences in endothelial cell states between the recipient cases were not statistically significant. The change in corneal endothelial cell density showed a significantly higher difference (p=0.0013) when patients had either undergone a rejection episode during recovery or recurred herpetic uveitis. CONCLUSIONS: The preoperative state of the corneal endothelium may affect the survival of donor corneal endothelium after grafting. However, rejection of the transplant contributes more significantly to the survival of the donor corneal endothelium than other factors. We suggest close observation and keen therapy with respect to rejection after grafting.

Two Cases of Corneal Ring Infiltrates from Topical Anesthetic Abuse

PURPOSE: Topical anesthetic abuse resulting in sight-threatening keratitis may be seen as a masquerade syndrome in many cases because of ring infiltration of the cornea. The authors report two cases of keratopathy from topical anesthetic abuse that were originally suspected as infectious keratitis because of ring infiltration of the cornea METHODS: The medical records of two patients were retrospectively reviewed. The pathogenesis, diagnosis, and treatment of ring infiltrates were evaluated. RESULTS: The two patients presented a nonhealing epithelial defect, marked stromal edema, folds in Descemet's membrane, and typical stromal ring infiltrates of unknown etiology. The patients initially were treated empirically with antibacterial and antifungal agents in suspicion of infectious keratitis. These patients had sustained severe ocular pain, and chronically used topical anesthetics (0.5% proparacaine hydrochloride in one patient and 0.4% benoxinate hydrochloride in the other) for several weeks. All microbiology work-ups for the identification of infectious organisms including acanthamoeba were negative. After topical anesthetic was discontinued in two patients, amniotic membrane transplantation was performed on one patient. Corneas in two patients were re-epithelialized with mild scarring with topical antibiotic and steroid treatment.

Corneal Ulcer and Chronic Conjunctivitis Due to Ofloxacin-resistant MRSA

PURPOSE: Considering popular use of fluoroquinolone eyedrops in Korea, it is important to know emerging resistant strain in treating infectious eye disease. We report methicillin resistant Staphylococcus aureus after frequent use of ofloxacin eyedrop in corneal ulcer and chronic conjunctivitis. METHODS: Four strains of ofloxacin-resistant MRSA (Methicillin-Resistant Staphylococcus Aureus) in patients with keratitis and conjunctivitis were found in our study. One strain was detected in a patient who used ofloxacin eyedrops intermittently for the treatment of recurrent corneal erosion which resulted from a bullous keratopathy after graft failure, and the others were detected in three patients using ofloxacin eyedrops intermittently or continuously for the treatment of conjunctivitis over 1 year. RESULTS: As fluoroquinolone eyedrop has been used more frequently, it is presumed that antibiotic resistance rate of ocular strains to ofloxacin might be increased. Therefore, a careful use of topical antibiotics based on the culture and antibiotic sensitivity test should be emphasized for the successful treatment of infectious ocular diseases.

A Comparison of Anti-Pseudomonal effects by Multi-purpose Contact Lens Solutions

PURPOSE: To compare the antimicrobial effects of various kinds of multi-purpose solutions, study effective ways of washing contact lens (CLs), and suggest the most effective lens care system using P. aeruginosa-contaminated CLs. METHODS: Each disposable soft CL was incubated in 0.1 ml of diluted solution of P. aeruginosa standardstrain (10(8)CFU / mm(3)) and 0.9 ml tryptic soy broth in cell culture wells for 24 hours. In the first experimental group, to find the most antimicrobial solution, 40 CLs contaminated by P. aeruginosa were divided into four subgroups washed with four different kinds of multi-purpose solutions. Ten CLs were used in each subgroup and in the control group; Ten CLs contaminated by P. aeruginosa were washed with phosphate buffered saline (PBS) instead of cleaning solution. In the second experiment groups to find the most effective way of washing, using only Renu Multiplus multi-purpose solutionR we compared the antimicrobial effect of four different ways of washing including 30 seconds rubbing, 30 minutes soaking, 4 hours soaking, 30 seconds rubbing and 4 hours soaking. RESULTS: There was no significant difference of antimicrobial effect between four kinds of multi-purpose solutions. All methods have a significant antimicrobial effect over control group (P<0.001) and the most effective method of washing CLs was 4 hours soaking. CONCLUSIONS: we should advise all the soft CL users that they should have their CLs soaked over 4 hours everyday after wearing it.

Endothelial Function of Cornea Preserved in Korean Corneal Storage Media

PURPOSE: To evaluate the endothelial function of cornea preserved in newly developing korean corneal storage media (CS002, CS003) by estimating the permeability of corneal endothelium and the change of corneal thickness. METHODS: The cornea were divided into six experimental groups - fresh group immediately after enucleation, 4degrees Cmoist chamber group preserved for 24 hours and 48 hours, Optisol & CS002 group for 1, 3, 5, and 7 days, and Likorol & CS003 group for 7, 10, and 14 days after enucleation, and then corneal endothelial permeability(Pac) was measured using carboxyfluorescein solution. Corneal thickness was measured using pachymeter(fine focus adjustment) of the specular microscope. RESULTS: Corneal endothelial Pac (x1 0(- 4) cm/min) was 3.64+/-0.33 in fresh group, 4.79+/-0.28 in 4degrees Cmoist chamber group for 24 hours. Each endothelial Pac of CS002 group at 5 and 7 days was 5.81+/-0.55 and 5.65+/-0.58, which were different with 4degrees Cmoist chamber preservation group for 24 hours(p<0.05) but not different with Optisol groups at same days. Each endothelial Pac of CS003 group at 7, 10, and 14 days was 4.34+/-0.34, 4.66+/-0.59, and 4.66+/-0.27, which were not different from those of Likorol. Each corneal thickness of CS002 and Optisol group at 7days was 417.80+/-19.37 mu m and 421.00+/-19.75mu m, which were resemble increment. Corneal thickness was 426.75+/-22.43mu m in CS003 group and 476.00+/- 40.08mu m in Likorol group at 7days. There was statistical difference between the two group(P<0.05), and this difference was sustained for 14days (P<0.05). CONCLUSION: There was no difference in the effect on corneal endothelial permeability between korean corneal storage media such as CS002 and CS003, and that of previous corneal storage media such as Optisol and Likorol. Corneal thickness of cornea preserved in korean corneal storage media was thinner than that of Likorol.

Correlation of sFas Level with Uveitis Severity

PURPOSE: The aim of this study is to determine the soluble Fas (sFas) levels in both sera and aqueous humor in patients with uveitis and compare them to the uveitis severity. METHODS: We measured the sFas levels in both sera and aqueous humor (AH) of patients (n=40) with uveitis and non-uveitis controls (n=27). The patients with uveitis comprised 24 Behcet's disease, 6 panuveitis, 5 anterior uveitis, 2 lens induced uveitis, 1 Vogt-Koyanagi-Harada-disease, 1 sarcoidosis, and 1 retinal vasculitis. The severity of uveitis was determined by the Hogan's grading method (0~4 grade) at the time of sampling. RESULTS:The concentration of aqueous sFas in uveitis patients was significantly higher than that in nonuveitis controls, while there was no difference in the serum concentration of sFas between the two groups. In the paired samples of serum and AH, obtained simultaneously, the aqueous sFas levels were higher than serum Fas levels in patients with uveitis, whereas the non-uveitis controls displayed significantly lower sFas levels in AH than in the serum. The sFas levels in AH or serum were not different between Behcet's uveitis and non-Behcet's uveitis. However, in patients with Behcet's uveitis, circulating sFas strongly correlated with aqueous sFas, which was not so in those with non-Behcet's uveitis. Patients (n=29) with more active (grade> or =2) uveitis had significantly higher levels of aqueous sFas than those (n=11) with less active (grade<2) uveitis. After treatment with steroid and/or immunosuppressive agents, aqueous sFas levels were decreased in parallel with a reduction in the number of inflammatory cells in the anterior chamber. CONCLUSIONS: The levels of sFas were elevated in patients with uveitis and correlated well with uveitis severity.

20% 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 Keratomileusis) instruments, and washed with sterile water. Zero time, 1, 3, 5 days after ethanol treatment, corneas were excised and examined with scanning and transmission electron microscopy. RESULTS: Widespread damage or disappearance of microvilli and local breaks of intercellular junction were observed. The changes were more severe in corneas with longer ethanol treament. In corneas with over 1 minute ethanol treatment, slough of superficial corneal epithelium was shown and increased with time. It was difficult to recognize microvilli or distinctive intercellular junction in corneas with 2 minute-treament. These pathologic changes persisted 5 days after ethanol-treatment. CONCLUSIONS: From these results, 30 seconds to 1 minute-ethanol treatment is recommended in corneal surgery to avoid severe, persisting damage of superficial corneal epithelium.

Efficacy of Polymerase Chain Reaction in Early Diagnosis of Herpes Simplex Keratitis in Rabbit Model

We evaluated the efficacy of polymerase chain reaction(PCR)in early diagnosis of herpes simplex keratitis(HSK)in rabbit model and compared the sensitivity and specificity of PCR in two kinds of primers. Only one eye of 8 rabbits was inoculated with HSV Type 1(F strain, ATCC VR-73)to induce HSK, and the other eye was used as control. Rabbit cornea was examined under slit lamp examination and PCR test of tear and corneal scraping specimens were performed at 1, 3, 5, 7, and 10th post-inoculation day. The sensitivity of PCR with POL primer was 100%in tear and corneal scraping specimens. The sensitivity of PCR with POL primer and LAT primer were 80%and 100%respectively. PCR test is very useful diagnotic tool for the early diagnosis of HSK in rabbit model. In addition, PCR test with corneal scraping specimens provided better yield compared with tears.

The Early Diagnosis of Herpetic Ke ratitis by Polymerase Chain Reaction

We evaluated the efficacy of polymerase chain reaction(PCR)in the early diagnosis of Herpes simplex virus keratitis(HSK). Among 47 patients(47 eyes), 25 patients were clinically herpetic keratitis and the others were other kinds of keratitis clinically. Tear film specimens were taken from 32 eyes and corneal scrapings from 21 eyes with suspicious herpetic keratitis and clinically incongrous with HSK. PCR for tear and corneal scraping was performed with using DNA primer. 67% of corneal scrapings and 19%tear samples of suspicious herpetic keratitis showed PCR positive. Tear samples from keratitis clinically incongruous with HSK were all PCR negative but 40%of corneal scrapings from these patients represented PCR positive. From these results, PCR is a rapid and effective tool for the early diagnosis of herpetic keratitis and especially useful in cases of nonspecific corneal lesion. However, for the accurate diagnosis of HSK, not only PCR results but also patient's history and corneal findings should be well evaluated.

Ocular Penetration of Topical Ofloxacin in Rabbit Eye with Amniotic Membrane Transplantation

PURPOSE: We evaluated ocular penetration and drug level in tear after topical ofloxacin instillation in rabbit eye with amniotic membrane transplantation(AMT). METHODS: In the first set of experiment, 24 rabbits(24 eyes) were divided into 4 groups according to the epithelial removal or AMT. Topical ofloxacin was instilled 4 times every 15 minutes and then, 1 hour after the last eyedrop, the samples of amniotic membrane(AM), cornea and aqueous humor were collected. In the second set of experiment, 24 rabbits(24 eyes) were divided into 6 groups according to the freshness of AM or its attached time. Topical ofloxacin was applied to eyes and then, tear samples were collected at 30 minutes, 1, 2, 4, and 6 hours. Ofloxacin concentration in the samples of two experiments were evaluated using high performance liquid chromatography. RESULTS: Mean ofloxacin concentrations in cornea and aqueous humor were statistically higher in deepithelized corneas regardless of AMT(P<0.05). And mean tear levels of ofloxacin in AMT groups were statistically higher than those in non-AMT groups(P<0.05). CONCLUSIONS: AMT seems to interfere with the ocular penetration of topical ofloxacin in normal rabbit cornea, but rather enhances ofloxacin penetration in the cornea with epithelial defect. And also the ofloxacin level in tear was higher in eyes with AMT up to 1 hour after topical ofloxacin use. Therefore it seems that AM has potential to act as an effective drug delivery system.

Epidemiology of Infectious Keratitis(II): A Multi-center Study

To identify risk factors and causative organisms, and to evaluate clinical manifestations, methods and results of treatment in infectious keratitis, an epidemiological study was performed prospectively under the identical protocol from April 1995 to March 2000.Logistic regression analysis was used to evaluate possible risk factors. The 1474 cases of infectious keratitis reported from 22 hospitals were studied. Five hundred forty-four organisms(442 bacteria, 82 fungi, 20 A c a n t h a m o e b a)were detected in 1320 eyes with infectious keratitis excluding 154 herpetic keratitis. The Pseudomonas aeruginosa was the most common organism in bacterial keratitis, and Fusariumspp. was the major isolate in fungal keratitis. Contact lens wear and occupation(industry, forester, miner, fisherman)were the risk factors for bacterial keratitis. Risk factors in fungal keratitis were fifth decade of age, farmer, and systemic diseases(diabetes mellitus etc.). Risk factors in herpetic keratitis were male and occupation(office worker, service, student, housewife). Risk factors in Acanthamoeba keratitis was contact lens wear.

Comparison of Antimicrobial Efficacy of Topical Antibiotics in Rabbit Keratitis Model with Ciprofl oxacin, Methicillin-Resistant Staphylococcus Aureus

To compare the antimicrobial efficacy of several antibiotics in the treatment of ciprofloxacin and methicillin-resistant staphylococcus aureus(MCRSA)keratitis, we established a rabbit keratitis model by using MCRSA isolated from keratitis patient. A strain of MCRSA, approroximately 100 colony forming units in 10 microliterof phosphate buffered saline was injected intrastromally into the each cornea of New Zealand white rabbits. 30 rabbtits(30 eyes)were randomly divided into 5 groups and treated with either 0.3%ciprofloxacin, 0.3%trovafloxacin, 5%vancomycin, 5%cefazolin, or sterile deionized water.Topical antibiotics were administered every 15 min for 6 hours(750microliter). Rabbits were sacrified 1hour after the last eyedrop, and excised corneas were homogenized, diluted, and plated on BAP medium.Bacterial colonyforming units per cornea were quantified after 48-hour incubation. Aqueous humor were collected from each eye and drug concentration was counted with HPLC. The log1 0 value of colony forming units(CFUs)of vancomycin treated group was 4.85+/-0.64;ciprofloxacin, 6.16+/-0.46;trovafloxacin, 6.58+/-0.29;cefazolin, 6.25+/-0.25;and sterile deionized water, 6.57+/-0.33.In view of mean CFU, vancomycin showed greater antimicrobial activity and it was statistically significant(p<0.05). However, all of these treatment did not completely sterilize any corneas with 6-hour treatment. From these results, only topical vancomycin(5%)represented effective antimicrobial activity with short-term treatment in rabbit keratitis with MCRSA.

B radykinin Receptor-Meadiated Intracellular Calcium Mobilization and ATP Changes in Cultured Bovine Corneal Endothelial Cell

To clarify the effect of bradykinin(Bk)on cultured bovine corneal endothelial cells(BCEC), cytosolic free calcium([Ca2+])mobilization and cell proliferation were investigated. The [Ca2+] was determined using a Ca2+ sensitive indicator, Fura-2/AM, and cell proliferation was evaluated by counting the cell number. Bk induced the transient increase of [Ca2+] in a concentration-dependent manner(10(-11)M~10(-7)M)and its 50% effective concentration was about 5x10(-11)M. The basal [Ca2+] with 1mM CaCl2 in the bathing solution was 87+/-9nM. Transient Bk(10(-8)M)-induced [Ca2+] increase was inhibited slightly but significantly by the pretreatment with EGTA. The pretreatment with U-73122(5x10(-6)M), an inhibitor of phospholipase C, also attenuated Bkinduced [Ca2+] mobilization. To identify and characterize the Bk receptor subtype in BCEC, Bk1 and Bk2 antagonists were investigated. Transient Bk(10(-8)M)-induced [Ca2+] increase was almost absolutely attenuated by the pretreatment with Bk2 antagonist for 10 minutes. To investigate the physiological effect of Bk, Bk-induced mitogenic effect was studied. 10(-8)M of Bk produced significant increase of intracellular ATP levels from the day 2 to the day six of culture period. This Bk-induced mitogenic effect was inhibited by the treatment with Bk2 antagonist. Bk-induced ion transport was determined by measuring intracellular ATP contents. Intracellular ATP content([ATP]i)was decreased by the treatment with 10(-8)M Bk for 10 minutes. Bk-induced [ATP]i decrement was significantly restored by the pretreatment with ouabain for 30 minutes. In summary, stimulation of intracellular signal transduction by Bk in BCEC is coupled with Bk2 type receptor. And also, Bk produces mitogenic effect and enhancesion and fluid transport in BCEC.

Morphologic Evaluation of Cat Corneal Endothelium Preserved in Korean Corneal Storage Medium

No Abstract Available.

Contamination of Contact Lens or Contact Lens Storage Case in Contact Lens Related Infectious Keratitis

In 44 out of 218 cases of contact lens related infectious keratitis from 19 hospitals throughout the country, contact lenses or contact lens storge cases were cultured. Microorganism was detected in 40 cases[90.9%]. Two or more organisms were isolated in 31 cases[77.5%]. Pseudomonas was the most common organism isolated from contact lens or contact lens storage medium[31 out of 84, 45.2%], followed by Serratia[15 out of 84, 17.9%], fungi [4], and acanthamoeba[4]. Acanthamoeba was found only in one hospital. Antibiotic sensitivity test for isolated pseudomonas showed that 96%of cases was sensitive to ciprofloxacin and 88%to ceftazidime.

Excimer Laser Photore fractive KeratectomyPRKfor Low and Mode rate Myopia using VIXS STAR Excimer Laser System

Excimer laser photorefractive keratectomy[PRK]for low and moderate myopia[-2D~-6D]has been performed recently as a predictable and effective method for correcting myopia. We analyzed refractive change, postoperative visual acuity, and postoperative complications of 168 eyes[104patients]for 6 months and 33 eyes among them for 1 year after excimer PRK using VISX STAR excimer laser system from February 1997 to February 1998.Among the patients male were 8 patients, and female were 96patients.The myopic range was from-2.00 diopters[D]to -6.00D with astigmatism less than 3D.Uncorrected visual acuity of 4/5 or better was achieved in 91%postoperatively ;corrected visual acuity of 4/5 or better in 97%postoperatively.Eighty-eight eyes received spherical photorefractive keratectomy[PRK]to correct myopia ;80 eyes received photoastigmatic refractive keratectomy[PARK]to correct both myopia and astigmatism.In spherical PRK group the mean spherical equivalent was -4.67 +/-1.69D preopratively, 0.11 +/-0.93D 6 months after PRK, -0.31 +/-0.80D 1 year after PRK ;in photoastigmatic refractive keratectomy[PARK]group these figures were -4 .9 4 +/-1.42D, -0.37 +/-0.81D, -0.62 +/-0.78D, respectively.In the PARK group13.8%were undercorrected but in the spherical PRK group only 5.7% were undercorrected.So we report the ndercorrection rate of PARK group was relatively higher than that of spherical PRK group, but statisticallyinsignificant[Chi-Square test, p=0.075, but Relative Risk=2.65].We evaluated the surgically induced astigmatism by using both Jaffe and Clayman's vector-corrected methods.In the spherical PRK group the mean surgically induced astigmatism[SIA]was 0.83 +/-0.49D 6 months after PRK, 0.72 +/-0.47D 1 year after PRK ;in the PARK group these figures were 1.36 +/-0.71D, 1.29 +/-0.72D, respectively.The differences between the two groups were statistically significant[unpaired T-test.p=0.001].Astigmatism greater than 0.25D was reduced in 23.86%and induced in 55.68%of eyes that received spherical PRK and astigmatism greater than 0.25D was reduced in 80%and induced in 3.75%of eyes that received PARK.There were several complications including transient punctate keratopathies[6 eyes, 3.5%], undercorrection [-1D][16 eyes, 9.5%], and overcorrection[-1D][28 eyes, 16.67%].The decrease in the best corrected visual acuity in 10 eyes[5.9%] may be the result of a grade 2 or more corneal haze[15 eyes]and central island[2 eyes].