Therapy for contact lens-related ulcers.

PURPOSE OF REVIEW: The current review covers the current literature and practice patterns of antimicrobial therapy for contact lens-related microbial keratitis (CLMK). Although the majority of corneal ulcers are bacterial, fungus and acanthamoeba are substantial contributors in CLMK and are harder to treat due to the lack of commercially available topical medications and low efficacy of available topical therapy. RECENT FINDINGS: Topical antimicrobials remain the mainstay of therapy for corneal ulcers. Fluoroquinolones may be used as monotherapy for small, peripheral bacterial ulcers. Antibiotic resistance is a persistent problem. Fungal ulcers are less responsive to topical medications and adjunct oral or intrastromal antifungal medications may be helpful. Acanthamoeba keratitis continues to remain a therapeutic challenge but newer antifungal and antiparasitic agents may be helpful adjuncts. Other novel and innovative therapies are being studied currently and show promise. SUMMARY: Contact lens-associated microbial keratitis is a significant health issue that can cause vision loss. Treatment remains a challenge but many promising diagnostics and procedures are in the pipeline and offer hope.

In Vivo Knockdown of the Herpes Simplex Virus 1 Latency-Associated Transcript Reduces Reactivation from Latency.

During herpes simplex virus (HSV) latency, most viral genes are silenced, with the exception of one region of the genome encoding the latency-associated transcript (LAT). This long noncoding RNA was originally described as having a role in enhancing HSV-1 reactivation. However, subsequent evidence showing that the LAT blocked apoptosis and promoted efficient establishment of latency suggested that its effects on reactivation were secondary to establishment. Here, we utilized an adeno-associated virus (AAV) vector to deliver a LAT-targeting hammerhead ribozyme to HSV-1-infected neurons of rabbits after the establishment of HSV-1 latency. The rabbits were then induced to reactivate latent HSV-1. Using this model, we show that decreasing LAT levels in neurons following the establishment of latency reduced the ability of the virus to reactivate. This demonstrates that the HSV-1 LAT RNA has a role in reactivation that is independent of its function in establishment of latency. In addition, these results suggest the potential of AAV vectors expressing LAT-targeting ribozymes as a potential therapy for recurrent HSV disease such as herpes stromal keratitis, a leading cause of infectious blindness.IMPORTANCE Herpes simplex virus (HSV) establishes a lifelong infection and remains dormant (latent) in our nerve cells. Occasionally HSV reactivates to cause disease, with HSV-1 typically causing cold sores whereas HSV-2 is the most common cause of genital herpes. The details of how HSV reactivates are largely unknown. Most of HSV's genes are silent during latency, with the exception of RNAs made from the latency-associated transcript (LAT) region. While viruses that make less LAT do not reactivate efficiently, these viruses also do not establish latency as efficiently. Here we deliver a ribozyme that can degrade the LAT to the nerve cells of latently infected rabbits using a gene therapy vector. We show that this treatment blocks reactivation in the majority of the rabbits. This work shows that the LAT RNA is important for reactivation and suggests the potential of this treatment as a therapy for treating HSV infections.

Adeno-associated Virus Vectors Efficiently Transduce Mouse and Rabbit Sensory Neurons Coinfected with Herpes Simplex Virus 1 following Peripheral Inoculation.

UNLABELLED: Following infection of epithelial tissues, herpes simplex virus 1 (HSV-1) virions travel via axonal transport to sensory ganglia and establish a lifelong latent infection within neurons. Recent studies have revealed that, following intraganglionic or intrathecal injection, recombinant adeno-associated virus (rAAV) vectors can also infect sensory neurons and are capable of stable, long-term transgene expression. We sought to determine if application of rAAV to peripheral nerve termini at the epithelial surface would allow rAAV to traffic to sensory ganglia in a manner similar to that seen with HSV. We hypothesized that footpad or ocular inoculation with rAAV8 would result in transduction of dorsal root ganglia (DRG) or trigeminal ganglia (TG), respectively. To test this, we inoculated the footpads of mice with various amounts of rAAV as well as rAAV capsid mutants. We demonstrated that this method of inoculation can achieve a transduction rate of >90% of the sensory neurons in the DRG that innervate the footpad. Similarly, we showed that corneal inoculation with rAAV vectors in the rabbit efficiently transduced >70% of the TG neurons in the optic tract. Finally, we demonstrated that coinfection of mouse footpads or rabbit eyes with rAAV vectors and HSV-1 resulted in colocalization in nearly all of the HSV-1-positive neurons. These results suggest that rAAV is a useful tool for the study of HSV-1 infection and may provide a means to deliver therapeutic cargos for the treatment of HSV infections or of dysfunctions of sensory ganglia. IMPORTANCE: Adeno-associated virus (AAV) has been shown to transduce dorsal root ganglion sensory neurons following direct intraganglionic sciatic nerve injection and intraperitoneal and intravenous injection as well as intrathecal injection. We sought to determine if rAAV vectors would be delivered to the same sensory neurons that herpes simplex virus (HSV-1) infects when applied peripherally at an epithelial surface that had been treated to expose the underlying sensory nerve termini. For this study, we chose two well-established HSV-1 infection models: mouse footpad infection and rabbit ocular infection. The results presented here provide the first description of AAV vectors transducing neurons following delivery at the skin/epithelium/eye. The ability of AAV to cotransduce HSV-1-infected neurons in both the mouse and the rabbit provides an opportunity to experimentally explore and disrupt host and viral proteins that are integral to the establishment of HSV-1 latency, to the maintenance of latency, and to reactivation from latency in vivo.

Overview of Corneal Transplantation for the Nonophthalmologist.

Corneal transplant is a procedure that aims to replace dysfunctional corneal tissue with a transparent graft and is one of the most widely performed transplant surgeries, but its public and professional awareness is low outside of ophthalmology. Corneal tissue consists of 5 major layers that serve to maintain its structural integrity and refractive shape: the epithelium, Bowman's layer, the stroma, Descemet's membrane, and the endothelium. Failure or irreversible damage to any layer of the cornea may be an indication for corneal transplant, and variants of this procedure may be full thickness or selectively lamellar. Complications related to corneal transplantation may occur anywhere from during surgery to years afterward, including rejection, dehiscence, cataract, and glaucoma. Complications should be managed by an ophthalmologist, but other physicians should be aware of prophylactic medications. Topical immunosuppressants and steroids are effective for preventing and treating rejection episodes, whereas there is little evidence to support the use of systemic immunosuppression. Eye protection is recommended for any corneal transplant recipient. Physicians should counsel patients on corneal donation, especially if outside the United States, where donor tissue is in short supply.

Reticular epithelial edema after penetrating keratoplasty in a patient taking netarsudil.

Netarsudil is a relatively new medication for the treatment of primary open-angle glaucoma and ocular hypertension. It has been associated with red eyes and burning after instillation. Reticular epitheliopathy is a relatively rare complication of netarsudil that has been described in patients with preexisting corneal edema. We report the case of a healthy 76-year-old woman who developed reticular epitheliopathy after full-thickness penetrating keratoplasty that completely resolved following discontinuation of the medication. In cases where netarsudil is initiated for treatment of glaucoma or, off-label, endothelial dysfunction, reticular epithelial edema should be considered in patients complaining of a decline in vision and severe pain.

Clinical and microbiological characteristics of fungal keratitis in the United States, 2001-2007: a multicenter study.

OBJECTIVE: To study the epidemiology, clinical observations, and microbiologic characteristics of fungal keratitis at tertiary eye care centers in the United States. DESIGN: Retrospective multicenter case series. PARTICIPANTS: Fungal keratitis cases presenting to participating tertiary eye care centers. METHODS: Charts were reviewed for all fungal keratitis cases confirmed by culture, histology, or confocal microscopy between January 1, 2001, and December 31, 2007, at 11 tertiary clinical sites in the United States. MAIN OUTCOME MEASURES: Frequency of potential predisposing factors and associations between these factors and fungal species. RESULTS: A total of 733 cases of fungal keratitis were identified. Most cases were confirmed by culture from corneal scraping (n = 693) or biopsies (n = 19); 16 cases were diagnosed by microscopic examination of corneal scraping alone; and 5 cases were diagnosed by confocal microscopy alone. Some 268 of 733 cases (37%) were associated with refractive contact lens wear, 180 of 733 cases (25%) were associated with ocular trauma, and 209 of 733 cases (29%) were associated with ocular surface disease. No predisposing factor was identified in 76 cases (10%). Filamentous fungi were identified in 141 of 180 ocular trauma cases (78%) and in 231 of 268 refractive contact lens-associated cases (86%). Yeast was the causative organism in 111 of 209 cases (53%) associated with ocular surface disease. Yeast accounted for few cases of fungal keratitis associated with refractive contact-lens wear (20 cases), therapeutic contact-lens wear (11 cases), or ocular trauma (21 cases). Surgical intervention was undertaken in 26% of cases and was most frequently performed for fungal keratitis associated with ocular surface disease (44%). Surgical intervention was more likely in cases associated with filamentous fungi (P = 0.03). Among contact lens wearers, delay in diagnosis of 2 or more weeks increased the likelihood of surgery (age-adjusted odds ratio = 2.2; 95% confidence interval, 1.2-4.2). CONCLUSIONS: Trauma, contact lens wear, and ocular surface disease predispose patients to developing fungal keratitis. Filamentous fungi are most frequently the causative organism for fungal keratitis associated with trauma or contact lens wear, whereas yeast is most frequently the causative organism in patients with ocular surface disease. Delay in diagnosis increases the likelihood of surgical intervention for contact lens-associated fungal keratitis.

Pseudodendritic fungal epithelial keratitis in an extended wear contact lens user.

PURPOSE: Pseudodendritic keratitis in a contact lens wearer is generally associated with acanthamoeba keratitis. We report a case of isolated pseudodendritic fungal epithelial keratitis that occurred in an extended wear contact lens user. METHODS: A 48-year-old woman was evaluated in our clinic for a 36-hour history of left eye pain. She wore extended wear soft contact lenses and frequently rinsed her eyes with tap water. Her left cornea had a paracentral 3-mm area of epithelium with raised ridges in a pseudodendritic pattern. The underlying corneal stroma was normal. A therapeutic and diagnostic corneal scraping of the lesion was performed and sent for Gomori methenamine silver (GMS) staining. The clinical concern was for epithelial acanthamoeba keratitis. RESULTS: The GMS staining revealed septate fungal hyphae within sheets of corneal epithelium. The patient was started on frequent alternating natamycin (5%) and amphotericin B (0.15%) antifungal eyedrops and exhibited a rapid clinical response. Her keratitis completely resolved, and her vision returned to her baseline of 20/25. Corneal fungal cultures showed no growth. CONCLUSIONS: Our case is an extremely unusual presentation of fungal keratitis, which rarely presents as a pseudodendritic epithelial keratitis. There are two previous similar case reports initially misdiagnosed as acanthamoeba keratitis. Clinicians should be aware that isolated fungal epithelial keratitis can present as a distinct entity and should be considered in the differential diagnosis of pseudodendritic keratitis. The GMS staining is an excellent diagnostic test in a patient presenting with pseudodendritic keratitis because it allows rapid diagnosis of acanthamoeba and fungal infections.

Trends in fungal keratitis in the United States, 2001 to 2007.

OBJECTIVE: Fungal keratitis is a serious ocular infection that is considered to be rare among contact lens wearers. The recent Fusarium keratitis outbreak raised questions regarding the background rate of Fusarium-related keratitis and other fungal keratitis in this population. DESIGN: Retrospective, multicenter case series. PARTICIPANTS: Six hundred ninety-five cases of fungal keratitis cases who presented to 1 of 10 tertiary medical centers from 2001 to 2007. METHODS: Ten tertiary care centers in the United States performed a retrospective review of culture-positive fungal keratitis cases at their centers between January 2001 and December 2007. Cases were identified using microbiology, pathology, and/or confocal microscopy records. Information was collected on contact lens status, method of diagnosis, and organism(s) identified. The quarterly number of cases by contact lens status was calculated and Poisson regression was used to evaluate presence of trends. The Johns Hopkins Medicine Institutional Review Board (IRB) and the IRBs at each participating center approved the research. MAIN OUTCOME MEASURES: Quarterly number of fungal keratitis cases and fungal species. RESULTS: We identified 695 fungal keratitis cases; 283 involved the use of contact lenses. The quarterly number of Fusarium cases increased among contact lens wearers (CLWs) during the period that ReNu with MoistureLoc (Bausch & Lomb, Rochester, NY) was on the market, but returned to prior levels after withdrawal of the product from the market. The quarterly frequency of other filamentous fungi cases showed a statistically significant increase among CLWs comparing October 2004 through June 2006 with July 2006 through December 2007 with January 2001 through September 2004 (P < 0.0001). CONCLUSIONS: The quarterly number of Fusarium fungal keratitis cases among CLWs returned to pre-Renu with Moistureloc levels after removal of the product from the market. However, the number of other filamentous fungal keratitis cases, although small, seems to have increased among refractive CLWs. Reasons for these apparent increases are unclear.

Reduction in severity of a herpes simplex virus type 1 murine infection by treatment with a ribozyme targeting the UL20 gene RNA.

Hammerhead ribozymes were designed to target mRNA of several essential herpes simplex virus type 1 (HSV-1) genes. A ribozyme specific for the late gene U(L)20 was packaged in an adenovirus vector (Ad-U(L)20 Rz) and evaluated for its capacity to inhibit the viral replication of several HSV-1 strains, including that of the wild-type HSV-1 (17syn+ and KOS) and several acycloguanosine-resistant strains (PAAr5, tkLTRZ1, and ACGr4) in tissue culture. The Ad-U(L)20 Rz was also tested for its ability to block an HSV-1 infection, using the mouse footpad model. Mouse footpads were treated with either the Ad-U(L)20 Rz or an adenoviral vector expressing green fluorescent protein (Ad-GFP) and then infected immediately thereafter with 10(4) PFU of HSV-1 strain 17syn+. Ad-U(L)20 ribozyme treatment consistently led to a 90% rate of protection for mice from lethal HSV-1 infection, while the survival rate in the control groups was less than 45%. Consistent with this protective effect, treatment with the Ad-U(L)20 Rz reduced the viral DNA load in the feet, the dorsal root ganglia, and the spinal cord relative to that of the Ad-GFP-treated animals. This study suggests that ribozymes targeting essential genes of the late kinetic class may represent a new therapeutic strategy for inhibiting HSV infection.

Delayed ectasia following LASIK with no risk factors: is a 300-microm stromal bed enough?

PURPOSE: To report a case of ectasia occurring > 4 years following LASIK with no risk factors and a residual stromal bed > 300 microm. METHODS: A 33-year-old woman presented 4 years after LASIK with mild blurring in the left eye. Uncorrected visual acuity (UCVA) had been 20/20 in both eyes previously. RESULTS: Uncorrected visual acuity was 20/20 and 20/40 in the right and left eyes, respectively. Best spectacle-corrected visual acuity (BSCVA) was 20/20 with -0.75 +2.25 x 70 degrees refraction in the left eye, which matched topography. Preoperative corneal thickness was 595 microm, and topography showed no risk factors preoperatively or immediately postoperatively. Calculated residual stromal bed was 342 microm and measured 400 microm with ultrasound microscopy. One year postoperatively, UCVA decreased to 20/400, and BSCVA decreased to 20/60 with refraction of -4.50 +5.00 x 90 degrees. The patient was intolerant of contact lens wear and is considering collagen cross-linking, Intacs, or corneal transplantation. CONCLUSIONS: Ectasia can occur more than 4 years after LASIK. Its etiology is unknown and management is challenging.

Shewanella putrefaciens keratitis in the lamellar bed 6 years after LASIK.

PURPOSE: To present a case of infectious keratitis occurring 6 years after LASIK due to the rare human pathogen Shewanella putrefaciens. METHODS: A 58-year-old man presented with redness and pain in the right eye 6 years following LASIK retreatment. Examination revealed a corneal infiltrate at the flap interface. Corneal scraping of stroma beneath the flap was submitted for histopathologic and microbiologic evaluation. RESULTS: An infiltrate located at the LASIK flap interface originated from an epithelial defect at the flap-corneal junction. Corneal stroma cultures demonstrated Shewanella putrefaciens. The infection resolved with antibiotic treatment. CONCLUSIONS: LASIK-related complications, such as infections, can occur many years following the procedure. The potential space created under the LASIK flap may predispose patients to infection by opportunistic organisms.

Science and strategy for preventing and managing corneal ulceration.

ABSTRACT Corneal ulcers can cause significant loss of vision from scarring and astigmatism, but rapid management can limit the destruction and improve outcomes. Infectious ulcers usually resolve with antimicrobial treatment. Noninfectious ulcers, however, present a diagnostic and therapeutic challenge. They can often be resolved by eliminating toxic medications and providing surface support with lubrication and collagenase inhibitors, but resistant ulcers may need more aggressive therapy with bandage contact lenses, tarsorrhaphy, or autologous serum. Ulcers impending perforation require urgent surgical management (e.g., tissue glue, conjunctival flaps, or keratoplasty). Topical steroids are useful when the ulceration is secondary to inflammatory mediators, but they are contraindicated in corneal melts with minimal inflammation, such as those associated with Sjogren syndrome. Systemic immunomodulation is required in addition to topical therapy in the presence of autoimmune disease. Understanding of the pathological processes that occur in different types of corneal ulcers is essential to formulation of a logical and effective treatment plan. Newer, more targeted treatment modalities may offer treatment options that have greater efficacy and fewer adverse effects.

Dual-Phase Iontophoresis for the Delivery of Antisense Oligonucleotides.

In support of ongoing research in the study of corneal and skin wound healing, we sought to improve on previously published results by using iontophoresis to deliver RNA interference-based oligonucleotides. By using a electromechanics-based approach, we were able to devise a two-phase solution that separated the buffering solution from the antisense oligonucleotide (ASO) solution. The separation was obtained by making the drug solution a higher density than the buffer, leading it to sink directly onto the tissue surface. This change immediately decreased the distance that the ASO would have to travel before delivery. The changes enabled delivery into ex vivo skin and corneas in 10 or fewer minutes and into in vivo corneas in 5 min. In vivo studies demonstrated short-term bioavailability of at least 24 h, a lack of chemical or thermal injury, a lack of interference in the healing of a corneal injury, and an antisense effect till at least day 7, but not day 14. The only side-effect observed was postdelivery edema that was not present when the vehicle alone was iontophoresed. This suggests that electro-osmotic flow from the delivery chamber was not the mechanism, but that the delivered solute likely increased the tissue's osmolarity. These results support the continued development and utilization of this ASO delivery approach in research-grade oligonucleotides to probe molecular biological pathways and in support of testing therapeutic ASOs in the skin and cornea.

Immunohistochemical localization of EGF, TGF-alpha, TGF-beta, and their receptors in rat corneas during healing of excimer laser ablation.

PURPOSE: Members of the epidermal growth factor (EGF) and transforming growth factor beta (TGF-beta) families of growth factors and receptors are known to regulate key aspects of corneal wound healing, including epithelial migration and scar formation. To further understand their roles, mRNA levels were measured and proteins were immunolocalized in rat corneas at multiple time points during healing of excimer laser ablation injury. METHODS: Excimer laser photoablation was performed to a depth of 50 microm on rat corneas. Levels of mRNAs for EGF, TGF-alpha, TGF-beta isoforms 1, 2, and 3, and their receptors (EGF-R and TGFbeta-IIR) were measured by quantitative RT-PCR on days 0, 1.5, 7, 21, 42, and 91 after ablation. Immunohistochemical localization of the growth factors and their receptors was performed on days 0, 7, and 21 in corneal sections. RESULTS: Levels of EGF mRNA remained stable in rat corneas after ablation (68 +/- 12 copies/cell, mean +/- SD), whereas levels of TGF-alpha mRNA progressively increased sixfold to a maximum at day 42 (300 copies/cell) then slightly decreased on day 91. Levels of EGF-R mRNA rapidly increased 60-fold on day 7 compared with day 0 (571 vs. 9 copies/cell) then decreased sixfold above baseline at day 91. Levels of TGF-beta1 mRNA remained stable (36 +/- 10 copies/cell), whereas levels of TGF-beta2 and TGF-beta3 mRNAs peaked on day 21 (300-fold and 25-fold increase) and remained elevated through day 91. Levels of TGFbeta-IIR mRNA showed a similar pattern. Immunostaining of all the growth factors and receptors was primarily in basal layers of epithelial cells in uninjured cornea and during healing. Intensity of immunostaining for TGF-beta1, TGFbeta-IR, and TGFbeta-IIR increased appreciably in the basal epithelial layers after ablation. CONCLUSIONS: Levels of mRNAs for several key members of the EGF and TGF-beta systems increase during corneal wound healing. In addition, the proteins are primarily localized in basal layers of epithelial cells, which suggest these cells are active in synthesizing autocrine and paracrine growth factors that modulate corneal wound healing.

Brothers with Smith-Lemli-Opitz syndrome.

Abnormal cholesterol metabolism is the cause of SLOS, with low cholesterol levels and elevated levels of cholesterol precursors thought to contribute to the clinical findings in this syndrome. Management of SLOS involves early intervention with appropriate therapies for identified disabilities, genetic counseling for families, nutritional consultations, educational interventions, and behavioral management. Although no randomized dietary studies have been conducted, cholesterol supplementation continues to be a common recommendation for persons with SLOS, because it may result in clinical improvement and has few adverse effects (Nowaczyk, 2013). Even with early detection and treatment (e.g., sibling B in this case report), persons with SLOS often have significant behavioral issues and cognitive and developmental delays that require a team approach by parents, educators, specialists, and primary care providers.

Connective tissue growth factor expression and action in human corneal fibroblast cultures and rat corneas after photorefractive keratectomy.

PURPOSE: Connective tissue growth factor (CTGF) has been linked to fibrosis in several tissues. In this study, the interactions between CTGF and transforming growth factor (TGF)-beta were assessed in human corneal fibroblasts, and the levels and location of CTGF protein and mRNA were measured during healing of excimer laser ablation wounds in rat corneas. METHODS: Human corneal fibroblasts were incubated with TGF-beta1, -beta2, and -beta3 isoforms, and CTGF mRNA and protein were measured. CTGF was immunolocalized in the cultured fibroblasts by using a specific antibody. Regulation of collagen synthesis by TGF-beta and CTGF was assessed in human corneal fibroblasts with a neutralizing antibody and an antisense oligonucleotide to CTGF. CTGF mRNA and protein were measured in rat corneas up to day 21 after excimer ablation of the cornea. CTGF protein was immunolocalized in rat corneas after photorefractive keratectomy (PRK), and the presence of CTGF mRNA and protein in ex vivo rat corneal scrapings was established. RESULTS: All three TGF-beta isoforms stimulated expression of CTGF in human corneal fibroblasts, and CTGF was immunolocalized in the cells. Both TGF-beta and CTGF increased collagen synthesis in corneal fibroblasts. Furthermore, CTGF antibody or antisense oligonucleotide blocked TGF-beta-stimulated collagen synthesis. CTGF protein and mRNA increased in rat corneas through day 21 after PRK. CTGF expression was also detected in ex vivo scrapings of rat corneas. CONCLUSIONS: These data demonstrate that CTGF is expressed by corneal cells after stimulation by TGF-beta, that CTGF expression increases significantly during corneal wound healing, and that CTGF mediates the effects of TGF-beta induction of collagen synthesis by corneal fibroblasts. These data support the hypothesis that CTGF promotes corneal scar formation and imply that regulating CTGF synthesis and action may be an important goal for reducing corneal scarring.

Curvularia keratitis after laser in situ keratomileusis from a feline source.

Fungal keratitis following laser in situ keratomileusis (LASIK) is extremely rare. Except in cases that follow trauma, the source of fungal organisms is unknown in published reports. We report a case of keratitis following LASIK that could be directly traced to a fungal skin infection of the patient's pet cat.