Herpetic Eye Disease Study: A Controlled Trial of Topical Corticosteroids for Herpes Simplex Stromal Keratitis.

PURPOSE: To evaluate the efficacy of topical corticosteroids in treating herpes simplex stromal keratitis. METHODS: The authors performed a randomized, double-masked, placebo-con- trolled, multicenter clinical trial of 106 patients with active herpes simplex stromal keratitis who had not received any corticosteroids for at least 10 days before study enrollment. Patients were assigned to the placebo group (n = 49) or the steroid group (topical prednisolone phosphate; n = 57); both regimens were tapered over 10 weeks. Both groups received topical trifluridine. Visual acuity assessment and slit-lamp biomicroscopy were performed weekly for 10 weeks, every other week for an additional 6 weeks or until removal from the trial, and at 6 months after randomization. RESULTS: The time to treatment failure (defined by specific criteria as persistent or progressive stromal keratouveitis or an adverse event) was significantly longer in the steroid group compared with the placebo group. Compared with placebo, corticosteroid therapy reduced the risk of persistent or progressive stromal keratouveitis by 68%. The time from randomization to resolution of stromal keratitis and uveitis was significantly shorter in the steroid group compared with the placebo group even though both groups included patients who were removed from the study and treated with topical corticosteroids according to best medical judgment. Nineteen (33%) of the steroid-treated patients and 11 (22%) of the placebo-treated patients completed the 10 weeks of protocol therapy and had stable, noninflamed corneas after 16 weeks. At 6 months after randomization, no clinically or statistically significant differences in visual outcome or recurrent herpetic eye disease were identified between the steroid and placebo groups. CONCLUSIONS: The topical corticosteroid regimen used in this study was significantly better than placebo in reducing persistence or progression of stromal inflammation and in shortening the duration of herpes simplex stromal keratitis. Postponing steroids during careful observation for a few weeks delayed resolution of stromal keratitis but had no detrimental effect as assessed by visual outcome at 6 months.

Dendritiform Keratopathy Associated with Exposure to Polyquarternium-1, a Common Ophthalmic Preservative.

PURPOSE: To describe dendritiform keratopathy associated with exposure to polyquaternium-1, a common preservative found in contact lens solutions and tear replacement products. DESIGN: Case series. PARTICIPANTS: Sixteen patients who demonstrated dendritiform keratopathy during topical ophthalmic exposure to polyquaternium-1. METHODS: Records were reviewed of all patients diagnosed with dendritiform keratopathy between 1999 and 2014 who had documented exposure to contact lens care disinfecting solutions or artificial tear solutions containing polyquaternium-1. Patients were excluded who had coexisting potential causes for dendritiform keratopathy, such as prior herpes simplex keratitis, varicella-zoster viral keratitis, the linear form of Thygeson's superficial keratitis, epithelial regeneration line, Acanthamoeba keratitis, mucus plaque keratopathy, medication-related keratopathy, or limbal stem cell deficiency characterized by conjunctivalization of the corneal epithelium. MAIN OUTCOME MEASURES: Effect of discontinuation of exposure to polyquaternium-1 on the dendritiform keratopathy. RESULTS: Sixteen patients demonstrated dendritiform keratopathy after exposure to the preservative polyquaternium-1. Thirteen patients had a history of recent exposure to contact lens disinfecting solutions (Opti-Free, Equate) containing polyquaternium-1. Three patients used a tear replacement product (Systane) containing a polyquaternium-1 preservative. Four patients were treated with antiviral medications for presumed herpes simplex keratitis; 4 patients underwent diagnostic testing for Acanthamoeba keratitis. Two additional patients were diagnosed sequentially with herpes simplex keratitis, then Acanthamoeba keratitis before referral. All dendritiform lesions resolved within 2 to 6 weeks after elimination of exposure to polyquaternium-1. CONCLUSIONS: Ophthalmic products containing polyquaternium-1 may cause dendritiform keratopathy that may be confused with infections of the superficial cornea, such as herpes simplex virus keratitis or Acanthamoeba keratitis.

Antiviral treatment and other therapeutic interventions for herpes simplex virus epithelial keratitis.

BACKGROUND: Eye disease due to herpes simplex virus (HSV) commonly presents as epithelial keratitis which, though usually self-limiting, may persist or progress without treatment. OBJECTIVES: To compare the relative effectiveness of antiviral agents, interferon, and corneal debridement in the treatment of HSV epithelial keratitis. SEARCH METHODS: We searched CENTRAL (which contains the Cochrane Eyes and Vision Group Trials Register) (2014, Issue 12), PubMed (January 1946 to 31 December 2014), EMBASE (January 1980 to 31 December 2014), Latin American and Caribbean Health Sciences Literature Database (LILACS) (January 1982 to 31 December 2014), System for Information on Grey Literature in Europe (OpenGrey) (January 1995 to 31 December 2014), BIOSIS (January 1926 to 5 May 2014), Scopus (January 1966 to 31 December 2014), Japan Science and Technology Institute (J-Global) (January 1975 to 31 December 2014), China National Knowledge Infrastructure (CNKI) (January 1979 to 31 December 2014), British Library's Electronic Table of Contents (Zetoc) (January 1993 to 7 May 2014). We looked for trials listed on the the metaRegister of Controlled Trials (www.controlled-trials.com), ClinicalTrials.gov (www.clinicaltrials.gov), the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP) (www.who.int/ictrp/search/en), Chinese Clinical Trial Registry, the U.S. Food and Drug Administration (FDA) (www.fda.gov/), National Institute for Health and Clinical Excellence (NICE) (www. EVIDENCE: nhs.uk) and the European Medicines Agency (EMA) (www.ema.europa.eu/ema/) as of 31 December 2014. There were no language or date restrictions in the search for trials. We also culled literature digests and conference proceedings as of 15 April 2014. There were no language or date restrictions in the search for trials. SELECTION CRITERIA: Randomised and quasi-randomised trials of HSV dendritic or geographic epithelial keratitis were included that reported the proportion of eyes healed at one week, two weeks, or both after enrolment. DATA COLLECTION AND ANALYSIS: We tabulated data on study characteristics, risk of bias, and outcomes and used direct comparisons to estimate a risk ratio (RR) and, when feasible, a hazard ratio (HR) with a 95% confidence interval (CI). Heterogeneity was assessed by an inconsistency index. A multiple treatment comparison meta-analysis consolidated direct and indirect comparisons of relative healing at 14 days. MAIN RESULTS: One hundred thirty-seven studies involving 8333 eyes met the inclusion criteria. Placebo-controlled studies were heterogeneous in comparison with idoxuridine (RR 1.74; 95% CI 1.03 to 2.91) and few in number for vidarabine (RR 1.81; 95% CI 1.09 to 3.01), interferon (RR 1.32; 95% CI 1.06 to 1.64), and debridement. Vidarabine (RR 1.13; 95% CI 1.02 to 1.25), trifluridine (RR 1.30; 95% CI 1.18 to 1.43), acyclovir (RR 1.23; 95% CI 1.14 to 1.34), and brivudine (RR 1.34; 95% CI 1.18 to 1.51) were more effective than idoxuridine. Trifluridine (RR 1.17; 95% CI 1.03 to 1.32) and acyclovir (RR 1.11; 95% CI 1.03 to 1.19) were more effective than vidarabine. No significant differences in healing emerged among trifluridine, acyclovir, brivudine, and foscarnet although few studies compared brivudine or foscarnet with other antivirals. Any potential advantage of ganciclovir compared to acyclovir was mitigated by study heterogeneity and possible publication bias. Only one study evaluated the joint use of two topical antivirals. In a limited number of studies, oral acyclovir (RR 0.92; 95% CI 0.79 to 1.07) or the combination of oral acyclovir with a topical antiviral (RR 1.36; 95% CI 0.68 to 2.74) appeared as effective as a single topical antiviral agent. Compared to topical antiviral monotherapy, the combination of an antiviral with either interferon or debridement had inconsistent effects on expediting healing and improving outcome. AUTHORS' CONCLUSIONS: Placebo-controlled studies of HSV epithelial keratitis are limited to superseded interventions. Trifluridine and acyclovir are more effective than idoxuridine or vidarabine and similar in therapeutic effectiveness. Brivudine and foscarnet do not substantially differ in effectiveness from trifluridine or acyclovir. Ganciclovir is at least as effective as acyclovir. The addition of interferon to a nucleoside antiviral agent and the combination of debridement with antiviral treatment need to be further assessed to substantiate any possible advantage in healing.

Ophthalmic viscosurgical device-assisted sutureless-incision cataract surgery for a hard nucleus or mature cataract.

UNLABELLED: We describe a modified method of sutureless-incision cataract surgery using an ophthalmic viscosurgical device (OVD) to extract a hard lenticular nucleus or mature cataract. After an isosceles trapezoid-shaped sclerocorneal tunnel and a round 7.0 mm diameter capsulorhexis are made, the nucleus is displaced into the anterior chamber. As OVD is injected, the OVD cannula acts as a slide to guide the nucleus out of the eye. Ophthalmic viscosurgical device-assisted sutureless-incision cataract surgery was used in a consecutive series of 182 eyes with a hard nucleus (57 eyes), mature cataract (47 eyes), or both (78 eyes). No posterior capsule rupture or vitreous loss occurred during surgery and no wound leakage or hypotony occurred postoperatively. The uncorrected visual acuity improved to 20/60 or better in 122 eyes (67%) on the first postoperative day. Ophthalmic viscosurgical device-assisted sutureless-incision cataract surgery, usually without additional instruments or sutures, offers an effective and uncomplicated technique for managing a brunescent or mature cataract. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Association between unprotected ultraviolet radiation exposure and recurrence of ocular herpes simplex virus.

Studies have suggested that exposure to ultraviolet (UV) light may increase risk of herpes simplex virus (HSV) recurrence. Between 1993 and 1997, the Herpetic Eye Disease Study (HEDS) randomized 703 participants with ocular HSV to receipt of acyclovir or placebo for prevention of ocular HSV recurrence. Of these, 308 HEDS participants (48% female and 85% white; median age, 49 years) were included in a nested study of exposures thought to cause recurrence and were followed for up to 15 months. We matched weekly UV index values from the National Oceanic and Atmospheric Administration to each participant's study center and used marginal structural Cox models to account for time-varying psychological stress and contact lens use and selection bias from dropout. There were 44 recurrences of ocular HSV, yielding an incidence of 4.3 events per 1,000 person-weeks. Weighted hazard ratios comparing persons with ≥8 hours of time outdoors to those with less exposure were 0.84 (95% confidence interval (CI): 0.27, 2.63) and 3.10 (95% CI: 1.14, 8.48) for weeks with a UV index of <4 and ≥4, respectively (ratio of hazard ratios = 3.68, 95% CI: 0.43, 31.4). Though results were imprecise, when the UV index was higher (i.e., ≥4), spending 8 or more hours per week outdoors was associated with increased risk of ocular HSV recurrence.

Corneal regrafting after endothelial keratoplasty.

PURPOSE: To determine the incidence of corneal regrafting after endothelial keratoplasty (EK) and to explore the possible reasons for repeat EK and subsequent penetrating keratoplasty (PK). METHODS: This retrospective cohort study examined the occurrence of corneal regrafts among 803 eyes of 751 patients who underwent initial EK from January 2004 through February 2009 using donor corneas distributed by a single eye bank. Regression models and life tables evaluated the effects of donor corneal characteristics on the probability of a regraft. RESULTS: Corneal regrafting after EK occurred in 119 eyes (15%), including 68 with repeat EK and 51 with subsequent PK. Ninety-five regrafts (80%) occurred within 1 year of EK, with 39 (33%) during the first postoperative month. Three years after EK, the cumulative probability of repeat EK was 11% and was 9% for subsequent PK. The secular trend in regrafting indicated an average 4% decline per year from 2005 to 2008. The odds of regrafting occurred less often (P = 0.004) with 202 eye bank-processed corneas than with 601 surgeon-prepared tissues. The cumulative probability of repeat EK was increased if donor corneas were maintained in preservation medium for more than 7 days (P = 0.02). Older donor age, death-to-preservation interval, or lower endothelial density was not significantly associated with repeat keratoplasty. CONCLUSIONS: Regrafting after EK is becoming less common, possibly because of surgical experience and technical innovations such as eye bank processing of precut tissues. Timely screening and distribution of donor corneas may foster graft survival.

Antiviral treatment and other therapeutic interventions for herpes simplex virus epithelial keratitis.

BACKGROUND: Eye disease due to herpes simplex virus (HSV) commonly presents as epithelial keratitis. OBJECTIVES: To compare the relative effectiveness of antiviral agents, interferon, and corneal débridement in the treatment of acute HSV epithelial keratitis. SEARCH STRATEGY: We searched CENTRAL (The Cochrane Library 2010, Issue 4), MEDLINE (January 1950 to October 2010), EMBASE (January 1980 to October 2010), Latin American and Caribbean Literature on Health Sciences (LILACS) (January 1982 to October 2010), Zetoc (British Library's Electronic Table of Contents), System for Information on Grey Literature in Europe (openSIGLE), Biosciences Information Service (BIOSIS), the metaRegister of Controlled Trials (mRCT) (www.controlled-trials.com), ClinicalTrials.gov (www.clinicaltrials.gov), Japan Information Center of Science and Technology (JICST-EPlus), and China Academic Journals database (CAJ) via China National Knowledge Infrastructure (CNKI) with citations confirmed using China/Asia On Demand (COAD). There were no language or date restrictions in the search for trials. All databases except CNKI and COAD were last searched on 27 October 2010, CNKI and COAD were searched on 1 April 2010. We also searched literature digests, conference proceedings and reference lists. SELECTION CRITERIA: Of 152 eligible studies,106 comparative treatment trials involving 5872 eyes with dendritic or geographic epithelial keratitis were analysed for corneal healing over two weeks. DATA COLLECTION AND ANALYSIS: Interventions were compared at 14 days after trial enrolment by calculating a risk ratio (RR) that was adjusted with indirect RR, assessed by an inconsistency index (I(2) ) and supplemented by a seven-day RR and a hazard ratio (HR). MAIN RESULTS: Idoxuridine, though uncertainly better in healing outcome than control because of few trials with 14-day follow up, allowed earlier corneal re-epithelialisation. Vidarabine resulted in a significantly better outcome than placebo in one trial (RR 1.96; 95% CI 1.10 to 3.49). Compared to idoxuridine, in combined direct and indirect analyses, vidarabine (RR 1.11; 95% CI 1.03 to 1.19), trifluridine (RR 1.31; 95% CI 1.20 to 1.42), acyclovir (RR 1.23; 95% CI 1.16 to 1.31), brivudine (RR 1.38; 95% CI 1.18 to 1.61), and ganciclovir (RR 1.40; 95% CI 1.25 to 1.57) were significantly more effective. Trifluridine (RR 1.12; 95% CI 1.04 to 1.21) and acyclovir (RR 1.11; 95% CI 1.05 to 1.19) appeared more effective than vidarabine. No significant differences were found in comparisons between acyclovir, trifluridine and brivudine. The comparison of ganciclovir to acyclovir was limited by heterogeneity and possible publication bias. The joint use of two topical antivirals (RR 1.00; 95% CI 0.89 to 1.12) and the use of oral acyclovir alone (RR 0.92; 95% CI 0.79 to 1.07) or combined with a topical antiviral (RR 1.08; 95% CI 0.99 to 1.17) appeared as effective as topical antiviral therapy. Compared to antiviral monotherapy, the combination of an antiviral with interferon (RR 1.03; 95% CI 0.99 to 1.07) or with débridement (RR 1.04; 95% CI 0.95 to 1.14) did not yield significantly better outcomes but may have accelerated healing. The corneal epithelial healing outcome was improved when antiviral therapy was added to débridement (RR 1.21; 95% CI 1.04 to 1.42). AUTHORS' CONCLUSIONS: Trifluridine and acyclovir are more effective than idoxuridine or vidarabine, and similar in therapeutic effectiveness. Brivudine and ganciclovir are at least as effective as acyclovir. While not improving outcome, the combination of interferon and an antiviral agent may speed healing. The effectiveness of corneal epithelial débridement is improved by an antiviral agent.

The molecular pathogenicity of Fusarium keratitis: a fungal transcriptional regulator promotes hyphal penetration of the cornea.

PURPOSE: The pathogenic mechanisms of fungal infection during human keratomycosis were investigated in an ex vivo corneal model that used strains of Fusarium oxysporum differing in the production of a fungal transcription factor. METHODS: A pacC loss-of-function mutant and a pacC dominant-activating mutant were constructed from a wild-type isolate of F. oxysporum, and the 3 strains were characterized by in vitro growth kinetics. Twenty-seven human donor corneas maintained in tissue culture were superficially scarified and topically inoculated with the wild-type, the pacC loss-of-function mutant, or the pacC dominant-activating strains. Relative hyphal invasion into the stroma was compared histopathologically in corneal sections. RESULTS: F. oxysporum strains demonstrated comparable exponential growth rates in vitro. Wild-type F. oxysporum invaded into the corneal tissue within 1 day and penetrated through the anterior stroma during the next 4 days. The pacC loss-of-function mutant invaded explanted corneas significantly less than the wild-type strain on day 1 (P < 0.0001) and on day 3 (P = 0.0003). The pacC dominant-activating strain adhered and penetrated explanted corneas similar to the wild-type strain. CONCLUSIONS: The PacC pathway regulating the transcription of fungal genes allows fungal adaptation to the ocular surface and enables invasion of the injured cornea by F. oxysporum.

The corneal expression of antimicrobial peptides during experimental fungal keratitis.

UNLABELLED: PURPOSE/AIM OF STUDY: To investigate the expression of endogenous antimicrobial peptides within the murine cornea during the onset and progression of posttraumatic keratomycosis caused by Candida albicans. MATERIALS AND METHODS: Scarified corneas of BALB/c mice were topically inoculated with C. albicans and monitored for one week. A murine gene microarray compared the relative expression of 36 antimicrobial peptide genes in infected corneas to controls. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) determined gene expression levels for murine cathelicidin and ß-defensins in normal corneas, scarified corneas, and C. albicans-infected corneas. Immunofluorescent staining localized the expression of cathelicidin in corneal sections. RESULTS: Traumatized eyes exposed to C. albicans developed progressive corneal inflammation, with a fungal inoculum of 10(6) colony-forming units (CFU) bringing about significantly (P < 0.05) more severe corneal inflammatory disease than a 10(5) CFU inoculum. Camp, encoding a murine cathelicidin-related antimicrobial peptide, was significantly upregulated 45-fold by microarray (P = 0.0007) and 36-fold by real-time RT-PCR (P = 0.0009). Camp increased significantly (P = 0.002) more in corneas receiving the higher than the lower fungal inoculum. Cathelicidin was preferentially expressed within the stroma on the first day after fungal inoculation, and Camp expression progressively declined over one week as the amount of recoverable fungi decreased. The genetic expression of ß-defensin 1 and ß-defensin 2 was initially downregulated (P ≤ 0.01) at the onset of fungal keratitis then returned toward normal levels. CONCLUSIONS: The antimicrobial peptide cathelicidin rapidly increases within the inflamed murine corneal stroma after the initiation of fungal keratitis and may play a role in the host responses that follow corneal trauma and infection.

Expression of small leucine-rich proteoglycans during experimental fungal keratitis.

PURPOSE: To investigate the expression of members of the small leucine-rich proteoglycan family and related leucine-rich repeat proteins during the inception and progression of experimental keratomycosis. METHODS: Scarified corneas of BALB/c mice were topically inoculated with Candida albicans and monitored daily over 1 week for corneal opacification. A murine gene microarray compared infected corneas to controls 1 day postinoculation (PI). Real-time reverse transcriptase polymerase chain reaction determined small leucine-rich proteoglycan gene levels in infected and mock-infected corneas at 1, 3, and 7 days PI and in normal corneas. Immunostaining localized keratocan protein in murine corneas. RESULTS: Eyes with C. albicans keratitis rapidly developed corneal inflammation with opacification. Microarray showed that genes for biglycan, asporin, lumican, fibromodulin, osteomodulin, keratocan, osteoglycin, and chondroadherin were significantly (P < 0.01) downregulated more than 2-fold at the onset of fungal keratitis. By real-time reverse transcriptase polymerase chain reaction, the gene encoding keratocan was initially downregulated 137-fold and remained downregulated 2.5-fold at 1 week. Genes coding for lumican, osteomodulin, and fibromodulin were downregulated 4- to 9-fold 1 day after fungal inoculation and returned to normal levels by 3 days PI. Immunofluorescence demonstrated that keratocan was present throughout the corneal stroma of normal mice and mock-infected controls but was markedly less during early fungal keratitis. CONCLUSIONS: Transcriptional levels of keratocan and other proteoglycans decrease during the initial stages of C. albicans keratitis. Alterations in the stromal extracellular matrix may contribute to the acute inflammatory response of corneal infection.

The RIM101 signal transduction pathway regulates Candida albicans virulence during experimental keratomycosis.

PURPOSE: To examine the role of the fungal RIM101 signal transduction pathway in the pathogenesis of Candida albicans keratitis. METHODS: C. albicans wild-type strain SC5314, prototrophic mutant control DAY185, and homozygous fungal mutants for the rim8, rim13, rim20, rim101, and phr1 genes were evaluated in vitro using proliferation and filamentation assays. Scarified corneas of BALB/c and C57BL/6J mice were topically inoculated and observed daily for keratitis severity. Corneal adaptation and pathogenicity were assessed ex vivo by maintaining infected porcine corneas for 3 days in an explantation culture system for histologic evaluation of hyphal penetration. RESULTS: All C. albicans strains had similar growth kinetics, and SC5314 and DAY185 demonstrated pH-induced filamentation. Fungal mutants had reduced hyphal formation at alkaline and neutral pH, but normal acidic assays ascertained that mutant strains did not have a generalized filamentation defect. SC5314 and DAY185 caused moderate to severe keratitis in mice, whereas fungal strains lacking constituents of the RIM101 pathway had significantly (P<0.05) attenuated severity in vivo. Three days after inoculation of porcine corneas, SC5314 and DAY185 produced hyphae that penetrated 28% and 25%, respectively, of the corneal thickness, and all five mutant strains showed significantly (P<0.05) less stromal penetration. CONCLUSIONS: The RIM101 signal transduction pathway plays an important role in the development of C. albicans keratitis. The fungal pathway intermediates Rim8p, Rim13p, Rim20p, and Rim101p and the downstream cell-wall protein Phr1p are pivotal in the process of corneal invasion by C. albicans.

Toll-like receptors involved in the pathogenesis of experimental Candida albicans keratitis.

Purpose. To investigate the expression and function of toll-like receptors (TLRs) during experimental keratomycosis. Methods. Scarified corneas of BALB/c mice were topically inoculated with Candida albicans and compared with control corneas by a murine gene microarray and immunostaining. Real-time reverse transcription polymerase chain reaction (RT-PCR) determined relative TLR gene expression in murine and human donor corneas. The scarified corneas of TLR2(-/-) mice, TLR4(-/-) mice, and C57BL/6J control mice were also inoculated with C. albicans, to determine relative severity, fungal load, and cytokine transcript levels. Results. TLR1, -2, -4, -6, and -13 were significantly upregulated (5- to 10-fold; P < 0.01) by microarray, and TLR1, -2, -4, and -13 were significantly increased (4- to 11-fold; P < 0.05) by real-time RT-PCR in BALB/c murine corneas. Similarly, TLR2, -6, and -13 were significantly upregulated (5- to 16-fold; P < or = 0.001) by real-time RT-PCR in C57BL/6J murine corneas the day after inoculation, and TLR2 and -13 remained significantly (P < 0.05) increased after 1 week. TLR2 transcript was also upregulated twofold (P = 0.04) in C. albicans-inoculated explanted human corneas. Although murine keratitis severity scores were similar, significantly more fungi were recovered from TLR2(-/-) mouse corneas (P = 0.04) than from TLR4(-/-) mouse corneas (P = 0.9). Tumor necrosis factor-alpha, interleukin 23, chemokine C-C ligands 3 and 4, and dectin-1 were significantly (P < 0.05) downregulated in C. albicans-infected corneas of TLR2(-/-) mice. Conclusions. TLR2 signals proinflammatory cytokines that control fungal growth during C. albicans keratitis. TLR13 may have an additional role in the innate immune response of murine corneal candidiasis.

A fungal pH-responsive signaling pathway regulating Aspergillus adaptation and invasion into the cornea.

PURPOSE: To investigate the role of PalB and PacC, two components of a pH-responsive signal-transduction pathway of Aspergillus nidulans, during the pathogenesis of fungal infection of the cornea. METHODS: Fungal strains included an A. nidulans wild-type isolate (A83), loss-of-function A. nidulans mutants of the palB (B7) or pacC (C6309) genes, and reconstituted genotypic strains (B7R and C6309R). Doubling times and radial growth rates were examined under neutral and acidic conditions. Corneal virulence was assessed ex vivo by topical inoculation of scarified porcine or human corneas with A. nidulans strains maintained in buffered medium until histologic examination after days 1, 3, and 5. RESULTS: In vitro growth kinetics were similar for A. nidulans strains in liquid medium at pH 6.0 (P = 0.24) and 7.3 (P = 0.75). The pacC mutant C6309 grew more slowly (P < 0.001) on solid medium, whereas palB and pacC rescuants had growth kinetics comparable to those of the wild-type. Wild-type A. nidulans germinated on porcine corneas and produced hyphae that progressively invaded the stroma, reaching an average maximum penetration of 56% +/- 9% at 5 days after exposure. In contrast, hyphal invasion was significantly less by mutant strains B7 (P = 0.005) and C6309 (P = 0.003). Fungal penetration by C6309 was also significantly less than the wild-type (P = 0.0005) on explanted human corneas. Both fungal rescuants showed stromal invasion similar to the wild-type. CONCLUSIONS: Corneal invasion by filamentous hyphae is attenuated by palB and pacC mutant strains of A. nidulans. The PacC pathway is involved in regulating fungal filamentation during ex vivo Aspergillus infection of the cornea.

Proinflammatory chemokines during Candida albicans keratitis.

Chemotactic cytokines mediate the recruitment of leukocytes into infected tissues. This study investigated the profile of chemokines during experimental Candida albicans keratitis and determined the effects of chemokine inhibition on leukocyte infiltration and fungal growth during murine keratomycosis. Scarified corneas of BALB/c mice were topically inoculated with C. albicans and monitored daily over one week for fungal keratitis. After a gene microarray for murine chemokines compared infected corneas to controls, real-time reverse transcription polymerase chain reaction (RT-PCR) and immunostaining assessed chemokine expression in infected and mock-inoculated corneas. An anti-chemokine antibody was then administered subconjunctivally and evaluated for effects on clinical severity, corneal inflammation, fungal recovery, and cytokine expression. Of 33 chemokine genes examined by microarray, 6 CC chemokines and 6 CXC chemokines were significantly (P<0.05) upregulated more than two-fold. Chemokine (CC-motif) ligand 3 (CCL3) was upregulated 108-fold (P=0.03) by real-time RT-PCR within one day after fungal inoculation and remained increased 28-fold (P=0.02) at one week, and its in situ expression increased in the epithelium and stroma of infected corneas. Compared to the control antibody-treated group, eyes treated with anti-CCL3 antibody showed reduced clinical severity (P<0.05), less corneal neovascularization (P=0.02), and fewer inflammatory cells infiltrating corneal tissue, but the amount of recoverable fungi was not significantly (P=0.4) affected. Anti-CCL3 treatment significantly (P=0.01) reduced the expression of tumor necrosis factor and interleukin-1beta in infected corneas. These results indicate that chemokines, especially the CC chemokine CCL3, play important roles in the acute inflammatory response to C. albicans corneal infection.

Corneal neovascularization during experimental fungal keratitis.

PURPOSE: To investigate the development of corneal neovascularization, the corneal expression of vascular endothelial growth factor (VEGF), and the antiangiogenic effects of a VEGF-inhibitory antibody during experimental keratomycosis. METHODS: Scarified corneas of BALB/c mice were topically inoculated with Candidaalbicans and monitored daily for corneal neovascularization. A murine gene microarray compared infected corneas to controls 1 day after inoculation. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) determined levels of genes encoding VEGF-A, VEGF-B, VEGF-C, and VEGF-D and placental growth factor in infected, mock-inoculated, and normal corneas. Immunostaining localized VEGF-A in corneal sections. An anti-VEGF-A antibody that binds to murine VEGF was evaluated for effects on corneal neovascularization and fungal recovery. RESULTS: Eyes with C. albicans keratitis manifested limbal capillary budding on the second postinoculation day, and intrastromal neovascular tufts subsequently grew at a mean rate of 250+/-80 microm/day. One day after the onset of C. albicans keratitis, VEGF-A was upregulated 12.5 fold (p=0.01) by microarray and 8.8 fold (p=0.004) by real-time RT-PCR, followed by a measured decline toward baseline over one week. VEGF-A was present in the epithelium and stroma of infected corneas. Scarification alone did not alter VEGF expression compared to the normal cornea. Anti-VEGF-A antibody significantly (p<0.01) decreased the formation of new corneal blood vessels during experimental keratomycosis without adversely affecting the fungal load of C. albicans keratitis. CONCLUSIONS: Untreated C. albicans keratitis induces VEGF-A and leads to progressive corneal neovascularization that is preventable by a VEGF-blocking antibody.

Morphogenic and genetic differences between Candida albicans strains are associated with keratomycosis virulence.

PURPOSE: To correlate the morphogenic and molecular traits that affect fungal virulence in human corneas. METHODS: C. albicans wild-type strains SC5314 and VE175 were compared using in vitro growth kinetics, filamentation assays, and microarray analysis. Corneal virulence was assessed ex vivo by inoculating C. albicans onto superficially scarified human corneas that were processed after 1 and 3 days to measure hyphal penetration. For comparison, DSY459, a C. albicans homozygous deletion mutant deficient in secreted aspartyl proteinases (SAP) 4, 5, and 6, was evaluated. RESULTS: C. albicans strain SC5314 was highly filamentous in vitro and more invasive in human corneal explants while VE175 demonstrated limited filamentation and less corneal invasion. Among 6,655 C. albicans genes, 9.0% significantly (p<.05) differed by 2 fold or more between SC5314 and VE175. Genes involved in fungal filamentation that were upregulated in strain SC5314 compared to VE175 included SAP5, SAP6, and other hypha-associated genes. Compared to wild-type strains, DSY459 had intermediate filamentation and stromal penetration. CONCLUSIONS: Fungal genes involved in filamentation likely contribute to virulence differences between wild-type strains of C. albicans. The corneal pathogenicity of C. albicans involves the morphogenic transformation of yeasts into hyphae.

Slitlamp biomicroscopy and photographic image analysis of herpes simplex virus stromal keratitis.

OBJECTIVE: To validate photographic bioimaging for evaluating the severity of herpes simplex virus keratitis. METHODS: Stromal keratitis of patients in the Herpetic Eye Disease Study was clinically measured with a slitbeam micrometer and then photographed at trial entry. Calibrated images of 169 eyes were analyzed for the size, location, and density of stromal keratitis and endotheliitis, with shape factor as a function of area and perimeter. Validity was assessed by comparing clinical and computerized measurements and by correlating the keratitis area with visual acuity. Logistic regression explored characteristics associated with larger or denser corneal inflammation. RESULTS: Stromal keratitis had a median area of 22.4 mm(2) (interquartile range, 12.8-31.6 mm(2)) with a median shape factor of 0.69 (interquartile range, 0.56-0.79); 126 eyes (75%) had their midpoint within 2 mm of the cornea's geometric center. Photoanalytical area estimates of herpetic stromal keratitis correlated closely with clinical measurements (correlation coefficient, 0.83). Eyes with larger stromal keratitis had worse vision (correlation coefficient, 0.32) and were more likely to have iritis (P = .01). Necrotizing stromal keratitis was significantly whiter (P = .02). CONCLUSIONS: Image analysis validly assesses the disciform geometry of herpetic stromal keratitis and confirms that increased severity is associated with uveitis and reduced vision.

Pathogenesis and outcome of Paecilomyces keratitis.

PURPOSE: To examine the clinical pathology and management of Paecilomyces lilacinus keratitis. DESIGN: Observational case series, literature review, and laboratory study. METHODS: Characteristics and outcome of 17 patients with laboratory-confirmed Paecilomyces keratitis treated at 2 referral centers were combined with 25 previously reported cases. Experimental models were developed by topically inoculating a human corneal isolate of P. lilacinus onto murine eyes and onto human donor corneas. RESULTS: Of 42 reported eyes with Paecilomyces keratitis, 13 (31%) were associated with chronic keratopathy or previous ocular surgery, 11 (26%) followed corneal trauma, and 10 (24%) occurred in soft contact lens wearers. Medical cure occurred in 13 (31%), including 9 of 31 eyes (29%) treated with natamycin or amphotericin B. Penetrating keratoplasty or other surgery was performed in 29 (69%). In vitro testing of P. lilacinus indicated resistance to natamycin and amphotericin B but susceptibility to ketoconazole and voriconazole. Experimental inoculation after superficial scarification established moderately severe corneal paecilomycosis by hyphae and conidia in immunosuppressed mice and in explanted donor corneas. CONCLUSIONS: P. lilacinus is an emerging fungal pathogen that infects corneal tissue by filamentous invasion with occasional intrastromal sporulation. P. lilacinus keratitis does not reliably respond to natamycin or amphotericin B and has often required therapeutic keratoplasty, but topical azole antifungal agents such as voriconazole appear promising.

Expression of matrix metalloproteinases during experimental Candida albicans keratitis.

PURPOSE: This study was designed to investigate the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) during the inception and progression of experimental keratomycosis. METHODS: Scarified corneas of adult BALB/c mice were topically inoculated with Candida albicans strain SC5314 and monitored for disease severity. Infected and mock-infected corneas were compared at 1 day post inoculation (p.i.) with a murine gene microarray. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) determined MMP and TIMP levels at 1, 3, and 7 days p.i. for infected, mock-infected, and normal corneas. Immunostaining localized target proteins at 1 day p.i. RESULTS: Eyes inoculated with C. albicans developed corneal infection with a mean clinical score of 8.2+/-0.8 at 1 day p.i. Compared to controls at 1 day p.i., MMP-8, -9, -10, -12, -13, -19, and TIMP-1 were significantly upregulated from fivefold to 375-fold by microarray and from threefold to 78-fold by real-time RT-PCR. Upregulated MMPs and TIMP-1 in the corneal epithelium and stroma of infected eyes correlated with the influx of acute inflammatory cells. Neither MMP-8 nor -13 expression was affected by mechanical trauma, but both increased >100-fold during the week after the onset of fungal keratitis. TIMP-1 expression rose from 21-fold more than controls at 1 day to 46-fold at 7 days p.i. by RT-PCR. CONCLUSIONS: Transcriptional and translational levels of MMP-8, -9, -13, and TIMP-1 increase during the early stages of C. albicans keratitis, confirming findings for MMP-9 and TIMP-1 in other infectious keratitis models and suggesting roles for MMP-8 and -13.