Investigating the Influence of Temperature and Supplementation Timing on Antifungal Efficacy in Storage Medium for Corneal Transplantation.

INTRODUCTION: Although antifungal supplementation reduces the fungal load in the corneal storage medium, consensus is lacking on the influence of dosing and temperature. The study aims to evaluate the impact of eye bank warming protocol and timing of antifungal supplements on efficacy in Optisol-GS and tissue. METHODS: Corneoscleral rims contaminated with Candida albicans (C. albicans) were incubated in Optisol-GS, either without antifungal agents or with the addition of amphotericin B or voriconazole at various concentrations (2 ×, 5 ×, 10 ×, and 20 × MIC), at different time points, and under various preservation temperatures (2-8 °C versus 2 h-room temperature exposure). Antifungal efficacy was evaluated by counting viable yeast colonies cultured from Optisol-GS samples. Tissue sterility was determined through direct tissue culture and histological examination of the contaminated rims after a 14-day incubation period. RESULTS: Room temperature exposure did not increase colony growth at the same multiple MIC of antifungal agents. Although antifungal addition reduced C. albicans growth in a concentration-dependent manner, yeast growth was still observed in all Optisol-GS samples with a single supplementation after a 14-day incubation. Only groups with additional antifungal supplementation on either day 2 or day 6 showed a 99% or greater reduction of C. albicans growth in Optisol-GS samples and yielded negative results in direct tissue culture. CONCLUSIONS: The eye bank warming protocol did not compromise antifungal efficacy. To sustain the required concentration and effectively reduce C. albicans growth in Optisol-GS and contaminated tissue, additional antifungal supplementation on either day 2 or day 6 was necessary during a 2-week preservation period.

Convolutional Neural Network-Based Prediction of Axial Length Using Color Fundus Photography.

Purpose: To develop convolutional neural network (CNN)-based models for predicting the axial length (AL) using color fundus photography (CFP) and explore associated clinical and structural characteristics. Methods: This study enrolled 1105 fundus images from 467 participants with ALs ranging from 19.91 to 32.59 mm, obtained at National Taiwan University Hospital between 2020 and 2021. The AL measurements obtained from a scanning laser interferometer served as the gold standard. The accuracy of prediction was compared among CNN-based models with different inputs, including CFP, age, and/or sex. Heatmaps were interpreted by integrated gradients. Results: Using age, sex, and CFP as input, the mean ± standard deviation absolute error (MAE) for AL prediction by the model was 0.771 ± 0.128 mm, outperforming models that used age and sex alone (1.263 ± 0.115 mm; P < 0.001) and CFP alone (0.831 ± 0.216 mm; P = 0.016) by 39.0% and 7.31%, respectively. The removal of relatively poor-quality CFPs resulted in a slight MAE reduction to 0.759 ± 0.120 mm without statistical significance (P = 0.24). The inclusion of age and CFP improved prediction accuracy by 5.59% (P = 0.043), while adding sex had no significant improvement (P = 0.41). The optic disc and temporal peripapillary area were highlighted as the focused areas on the heatmaps. Conclusions: Deep learning-based prediction of AL using CFP was fairly accurate and enhanced by age inclusion. The optic disc and temporal peripapillary area may contain crucial structural information for AL prediction in CFP. Translational Relevance: This study might aid AL assessments and the understanding of the morphologic characteristics of the fundus related to AL.

The versatile roles of lumican in eye diseases: A review.

Lumican is a keratan sulfate proteoglycan that belongs to the small leucine-rich proteoglycan family. Research has lifted the veil on the versatile roles of lumican in the pathogenesis of eye diseases. Lumican has pivotal roles in the maintenance of physiological tissue homogenesis and is often upregulated in pathological conditions, e.g., fibrosis, scar tissue formation in injured tissues, persistent inflammatory responses and immune anomaly, etc. Herein, we will review literature regarding the role of lumican in pathogenesis of inherited congenital and acquired eye diseases, e.g., cornea dystrophy, cataract, glaucoma and chorioretinal diseases, etc.

Maximize Donor Cornea Use in a Hepatitis B Endemic Area via Serology Matching.

BACKGROUND: This study aims to investigate the rationality of the allocation guidelines in a hepatitis B endemic area that uses corneas from hepatitis B donors. METHODS: Under Taiwan's current guidelines, corneas donated from hepatitis B surface antigen (HBsAg)(+) donors can be allocated to HBsAg(+) or hepatitis B surface antibody recipients. From January 1, 2015, to December 31, 2019, corneas donated to National Taiwan University Hospital were divided into HBsAg(+), HBsAg(-)/hepatitis B core antibody (anti-HBc)(+), and HBsAg(-)/anti-HBc(-) groups. Hepatitis B virus (HBV) DNA extracted from corneoscleral rims was quantified by polymerase chain reaction and correlated with donor serum HBsAg, anti-HBc, and HBV DNA. Recipients of HBV DNA(+) grafts were called back for serology and serum HBV DNA tests. RESULTS: The corneoscleral HBV DNA of 170 corneas (113 donors) was quantified, of which 45 corneas were from 28 HBsAg(+) donors, 87 were from 57 HBsAg(-)/anti-HBc(+) donors, and 38 were from 28 HBsAg(-)/anti-HBc(-) donors, and HBV DNA was detected in 80.0%, 9.2%, and 0% of the corneoscleral rims in each group. Donor anti-HBc(+) provided the highest sensitivity (1.00) and negative predictive value (1.00) for corneoscleral HBV DNA. Twenty-eight of 40 recipients (70%) using HBV DNA(+) grafts were called back, and none developed hepatitis in follow-up periods ranging from 6 to 55.5 mo. CONCLUSIONS: Donor anti-HBc should be tested routinely with HBsAg. Allocating corneas from HBsAg(+) or anti-HBc(+) donors to HBsAg(+) or hepatitis B surface antibody recipients maximizes cornea usage from hepatitis B donors without compromising transplant safety in a hepatitis B endemic setting.

Keratoplasty Registries: Lessons Learned.

ABSTRACT: Clinical registries have been developed for decades in the field of ophthalmology, and they are especially well-suited to the study of keratoplasty practices. A comprehensive donor/recipient registry system can provide insight into donor, recipient, and surgical factors associated with immediate and long-term outcomes and adverse reactions. Furthermore, linkage with demographic databases can elucidate relationships with social determinants of health and potentially shape public policy. The vast sample size and multicenter nature of registries enable researchers to conduct sophisticated multivariate or multilayered analyses. In this review, we aim to emphasize the importance of registry data for keratoplasty practice and 1) summarize the structure of current keratoplasty registries; 2) examine the features and scientific contributions of the registries from Australia, the United Kingdom, Singapore, the Netherlands, Sweden, Eye Bank Association of America, and European Cornea and Cell Transplant registries; 3) compare registry-based studies with large single-site clinical studies; 4) compare registry-based studies with randomized control studies; and 5) make recommendations for future development of keratoplasty registries. Keratoplasty registries have increased our knowledge of corneal transplant practices and their outcomes. Future keratoplasty registry-based studies may be further strengthened by record linkage, data sharing, and international collaboration.

Extending the utility of anterior corneal buttons through refrigeration and glycerol cryopreservation: utility rate and outcome analysis.

BACKGROUND/AIMS: To evaluate the utility rate, indication, outcome, and cost of refrigeration and glycerol cryopreservation for storing anterior corneal buttons during endothelial keratoplasty for subsequent use in tectonic lamellar patch grafting. METHOD: Anterior corneal buttons collected after precutting or prestripping during endothelial keratoplasty from January 2014 to December 2019 were preserved using the following protocol: (1) refrigeration for up to 4 weeks at 4°C in Optisol-GS and (2) glycerol cryopreservation for up to 2 years. The utility rate, outcome and cost of these cryopreserved anterior corneal buttons were retrospectively examined. RESULTS: During the 6-year study period, 26 anterior corneal buttons were refrigerated and 49 were cryopreserved for extended use. The utility rates for the refrigerated and cryopreserved anterior corneal buttons were 69.2% and 73.5%, respectively. Their average preservation periods were 0.53±0.05 and 12.76±0.94 months, respectively. Noninfection-related perforation was the leading indication for using the extendedly preserved anterior corneal buttons. The average postoperative follow-up periods were 10.03±2.91 and 14.35±2.17 months for refrigerated and cryopreserved anterior corneal buttons. Secondary keratoplasty was required by 7 of 18 (38.9%) and 6 of 36 (16.7%) patients receiving refrigerated and cryopreserved anterior corneal buttons, respectively. None of our patients developed graft infection from donor tissues. CONCLUSION: Cryopreservation can safely extend the utility of anterior corneal buttons. This method not only reduced the wastage of the limited donor tissue but also was cost-effective.

Integrated Stress Response Regulation of Corneal Epithelial Cell Motility and Cytokine Production.

Purpose: To investigate the effect of an active integrated stress response (ISR) on human corneal epithelial cell motility and cytokine production. Methods: ISR agonists tunicamycin (TUN) and SAL003 (SAL) were used to stimulate the ISR in immortalized corneal epithelial cell lines, primary human limbal epithelial stem cells, and ex vivo human corneas. Reporter lines for ISR-associated transcription factors activating transcription factor 4 (ATF4) and XBP1 activity were generated to visualize pathway activity in response to kinase-specific agonists. Scratch assays and multiplex magnetic bead arrays were used to investigate the effects of an active ISR on scratch wounds and cytokine production. A C/EBP homologous protein (CHOP) knockout cell line was generated to investigate the effects of ISR ablation. Finally, an ISR antagonist was assayed for its ability to rescue negative phenotypic changes associated with an active ISR. Results: ISR stimulation, mediated through CHOP, inhibited cell motility in both immortalized and primary human limbal epithelial cells. Scratch wounding of ex vivo corneas elicited an increase in the ISR mediators phosphorylated-eIF2α and ATF4. ISR stimulation also increased the production of vascular endothelial growth factor (VEGF) and proinflammatory cytokines. ISR ablation, through CHOP knockout or inhibition with integrated stress response inhibitor (ISRIB) rescued epithelia migration ability and reduced VEGF secretion. Conclusions: We demonstrate that the ISR has dramatic effects on the ability of corneal epithelial cells to respond to wounding models and increases the production of proinflammatory and angiogenic factors. Inhibition of the ISR may provide a new therapeutic option for corneal diseases in which the ISR is implicated.

Center-for-Near Extended-Depth-of-Focus Soft Contact Lens for Myopia Control in Children: 1-Year Results of a Randomized Controlled Trial.

INTRODUCTION: This study aimed to assess the safety and efficacy of a novel extended-depth-of-focus (EDOF) soft contact lens for myopia control in children. METHODS: A prospective, multicenter, randomized, double-masked, placebo-controlled, contralateral-eye comparison clinical trial was conducted in 72 children (40 male and 32 female) aged 9 to 14 years, with each eye randomly selected to wear either an experimental EDOF contact lens or a single-vision control lens at least 8 h per day, 5 days a week, for 52 weeks. Each contact lens was worn and then replaced daily. Measurements including best-corrected visual acuity, spherical equivalent refractive error (SER), axial length (AXL), and keratometry were performed at weeks 1, 4, and 13, and every 13 weeks thereafter for 52 weeks. The primary outcome measure was the change in SER, measured using cycloplegic auto-refraction. The secondary outcome measure was the change in AXL. RESULTS: At week 52, the mean change in SER was significantly lower with the experimental lens (-0.70 ± 0.49 D) than with the control lens (-0.88 ± 0.51 D; P < .001). The mean AXL elongation was significantly lower with the experimental lens (0.34 ± 0.19 mm) than with the control lens (0.38 ± 0.19 mm; P < .001). The EDOF lens reduced AXL and myopia progression by 10.5% and 20.5%, respectively. The change in SER, but no AXL, was significantly associated with EDOF lens wear in adjusted multivariate regression analysis. Reported adverse events did not differ significantly between the two lens types. CONCLUSIONS: The results of this 1-year clinical trial demonstrate that the experimental EDOF soft contact lens slows myopia progression and reduces AXL elongation in children compared with a single-vision contact lens. (This study was retrospectively registered with ClinicalTrials.gov; identifier: NCT04238897; date of registration: January 23, 2020.).

Toxic keratopathy related to antiseptics in nonocular surgery.

Antiseptics, especially those containing ethanol, are toxic to the ocular surface. Here, we report a 5-year-old girl with antiseptic-related eye injury following an uneventful bilateral tonsillectomy under general anesthesia. Before surgery, her eyes were protected and disinfection of perioral skin with ethanol-containing chlorhexidine followed. Whitening of the lower half of her right ocular surface was found after the surgery, and this indicated severe chemical burn. Prompt irrigation with normal saline, instillation of topical medication, and application of amniotic membrane containing device were performed, which led to a satisfactory result. Toxic eye injury could happen in head and neck surgeries under general anesthesia. Causes of ocular injury include improper eye protection, head positions leading to accumulation of excessive antiseptics, and improper usage of ethanol-containing antiseptics for skin preparation. The use of ethanol-free antiseptic solutions in the peri-ocular region and proper protection of eyes may reduce the risk of severe ocular surface injury in nonocular surgeries.

Review, analysis, and education of antiseptic related ocular injury in the surgical settings.

Preoperative skin antiseptic preparation is the gold standard for prevention of surgical infection. However, improper use of antiseptics may lead to severe ocular damage. Currently, the most common surgical antiseptics can be divided into aqueous-based and alcohol-based disinfectants, with chlorhexidine and iodine/iodophors being the two major components. Chlorhexidine has a persistent antimicrobial effect and is resistant to neutralization by blood or organic products in surgical wounds. Nevertheless, due to its toxicity to the ears, meninges, and eyes, application of chlorhexidine should be prohibited in these surgical fields. Iodine/iodophor is better tolerated by the ocular surface and is the recommended antiseptic for ophthalmic or head and neck surgeries close to the periocular area. Alcohol is less pricey and has a rapid antiseptic effect, though its desiccating effect and flammability restrict the use in mucosal or laser surgeries. The single or combined use of these antiseptics may inadvertently induce severe ocular damage, especially during time-consuming head and neck surgeries with prone, hyperextension, or lateral tilt positions, or surgeries under general anesthesia. Apart from the choice of antiseptics, appropriate selection and attachment of bio-occlusive dressings are key to avoiding antiseptic-related ocular injuries. In this review, we provided a comprehensive summary of the characteristics of antiseptics used in surgical settings and the possible mechanisms and outcomes of antiseptic-related ocular injuries. The prevention, diagnosis, and acute management of these complications were also discussed.

Spectral-domain optical coherence tomography for evaluating palisades of Vogt in ocular surface disorders with limbal involvement.

Spectral-domain optical coherence tomography (SD-OCT) has been used to observe the morphology of the palisades of Vogt (POV) with satisfactory resolutions. In this study, we used SD-OCT to examine the microstructure of the POV in ocular surface disorders with limbal involvement. We detect subclinical limbal pathologies based on five parameters, including (1) decreased epithelial thickness, (2) loss of the sharp stromal tip, (3) loss of the smooth epithelial-stromal interface, (4) dilated stromal vessels, and (5) decreased POV density. Eighteen eyes of 10 patients with advancing wavelike epitheliopathy (AWE) and 15 eyes of 9 patients with phlyctenular keratitis/ocular rosacea were recruited. SD-OCT could detect abnormal changes in the POV in 100% of the lesion sites. In presumed-healthy areas of the diseased eyes diagnosed by slit-lamp biomicroscopy, SD-OCT detected abnormal changes in the POV in 100% of the eyes in both groups. In patients with unilateral disease, abnormal changes in the POV were detected by SD-OCT in 50% and 100% of presumed-healthy eyes diagnosed by slit-lamp biomicroscopy in the AWE group and phlyctenular keratitis/ocular rosacea group, respectively. SD-OCT is powerful in detecting POV changes in ocular surface disorders and can provide useful information that cannot be provided by slit-lamp biomicroscopy.

Submicron spatial resolution optical coherence tomography for visualising the 3D structures of cells cultivated in complex culture systems.

Three-dimensional (3D) configuration of in vitro cultivated cells has been recognised as a valuable tool in developing stem cell and cancer cell therapy. However, currently available imaging approaches for live cells have drawbacks, including unsatisfactory resolution, lack of cross-sectional and 3D images, and poor penetration of multi-layered cell products, especially when cells are cultivated on semitransparent carriers. Herein, we report a prototype of a full-field optical coherence tomography (FF-OCT) system with isotropic submicron spatial resolution in en face and cross-sectional views that provides a label-free, non-invasive platform with high-resolution 3D imaging. We validated the imaging power of this prototype by examining (1) cultivated neuron cells (N2A cell line); (2) multilayered, cultivated limbal epithelial sheets (mCLESs); (3) neuron cells (N2A cell line) and mCLESs cultivated on a semitransparent amniotic membrane (stAM); and (4) directly adherent colonies of neuron-like cells (DACNs) covered by limbal epithelial cell sheets. Our FF-OCT exhibited a penetrance of up to 150 µm in a multilayered cell sheet and displayed the morphological differences of neurons and epithelial cells in complex coculture systems. This FF-OCT is expected to facilitate the visualisation of cultivated cell products in vitro and has a high potential for cell therapy and translational medicine research.

Targeting the integrated stress response in ophthalmology.

Purpose: To summarize the Integrated Stress Response (ISR) in the context of ophthalmology, with special interest on the cornea and anterior segment. Results: The ISR is a powerful and conserved signaling pathway that allows for cells to respond to a diverse array of both intracellular and extracellular stressors. The pathway is classically responsible for coordination of the cellular response to amino acid starvation, ultraviolet light, heme dysregulation, viral infection, and unfolded protein. Under normal circumstances, it is considered pro-survival and a necessary mechanism through which protein translation is controlled. However, in cases of severe or prolonged stress the pathway can promote apoptosis, and loss of normal cellular phenotype. The activation of this pathway culminates in the global inhibition of cap-dependent protein translation and the canonical expression of the activating transcription factor 4 (ATF4). Conclusion:The eye is uniquely exposed to ISR responsive stressors due to its environmental exposure and relative isolation from the circulatory system which are necessary for its function. We will discuss how this pathway is critical for the proper function of the tissue, its role in development, as well as how targeting of the pathway could alleviate key aspects of diverse ophthalmic diseases.

Pigment Epithelium-Derived Factor Peptide Promotes Corneal Nerve Regeneration: An In Vivo and In Vitro Study.

Purpose: To investigate the potential of a pigment epithelium-derived factor (PEDF) peptide 44-mer to promote nerve regeneration in a rabbit corneal nerve injury model to demonstrate its neurotrophic ability in cultivated mouse trigeminal neuron cells. Methods: Subconjunctival or intrastromal injection of 44-mer on the cornea was performed in a rabbit model of corneal nerve injury created by corneal epithelial debridement. Immunocytochemical analysis (44-mer, anti-tubulin III, SMI312, CD11b, and α-SMA) and in vivo confocal microscopy were performed. Corneal sensation was estimated using a Cochet-Bonnet corneal esthesiometer. Primary cultivated mouse trigeminal neurons were used to examine the in vitro neurotrophic ability of 44-mer. The cellular morphology and the immunocytochemical staining with anti-tubulin III and SMI312 in different concentrations of 44-mer were compared, and a quantitative assessment of neurite outgrowth was performed. Results: Immunohistochemical staining showed the retention of 44-mer in the corneal stroma for at least 7 days after a single dose of corneal intrastromal injection and promoted corneal nerve regeneration revealed by in vivo confocal microscopy. Corneal esthesiometer demonstrated gradual recovery of the corneal sensation in 44-mer-treated eyes with a lower corneal touch threshold than wounded vehicles and closer to baseline at 3 weeks after corneal injury (P < 0.001). In vitro studies showed a dose-dependent neurotrophic effect of 44-mer in cultivated trigeminal neuron cells. Conclusions: The 44-mer showed in vivo and in vitro corneal neurotrophic abilities. Our results suggest that intrastromal injection of 44-mer into the corneal stroma may have a potential role in treating diseases related to corneal nerve damage.

The effect of human platelet lysate on corneal nerve regeneration.

AIM: This study aimed to test whether human platelet lysate (HPL) has neurotrophic ability for corneal nerve regeneration. METHODS: We measured the neurotrophic factors in human peripheral serum (HPS) and two commercially available HPLs, UltraGRO and PLTMax. In vitro, we compared the growth rates, neuronal differentiation and immunostaining of neuron markers in mouse neuroblastoma cell line (Neuro-2a) and primary culture of mouse trigeminal ganglion cells that were cultivated in different concentrations of fetal bovine serum, HPS and HPL. In vivo, we created corneal wounds on Sprague Dawley rats with a rotating burr and evaluated the effects of topical HPL on wound healing and corneal nerve regeneration by in vivo confocal microscopy and corneal aesthesiometry. RESULTS: HPLs had significantly higher concentrations of various neurotrophic factors compared with HPS (p<0.05). In Neuro-2a cells, 3% HPL was better at promoting neuronal growth and differentiation compared with HPS at the same concentration. HPL was also found to have superior neurotrophic effects compared with HPS in primary cultures of mouse trigeminal ganglion cells. In vivo, HPL-treated eyes had better corneal epithelial wound healing rate, nerve regeneration length and corneal touch threshold compared with eyes treated with artificial tears (p<0.05). CONCLUSION: HPL has significantly higher concentrations of neurotrophic factors compared with HPS. It showed not only in vitro but also in vivo corneal neurotrophic abilities. Our results suggest that HPL may have a potential role in the treatment of diseases related to corneal nerve damage or degeneration.

Keratoconus Screening Based on Deep Learning Approach of Corneal Topography.

Purpose: To develop and compare deep learning (DL) algorithms to detect keratoconus on the basis of corneal topography and validate with visualization methods. Methods: We retrospectively collected corneal topographies of the study group with clinically manifested keratoconus and the control group with regular astigmatism. All images were divided into training and test datasets. We adopted three convolutional neural network (CNN) models for learning. The test dataset was applied to analyze the performance of the three models. In addition, for better discrimination and understanding, we displayed the pixel-wise discriminative features and class-discriminative heat map of diopter images for visualization. Results: Overall, 170 keratoconus, 28 subclinical keratoconus and 156 normal topographic pictures were collected. The convergence of accuracy and loss for the training and test datasets after training revealed no overfitting in all three CNN models. The sensitivity and specificity of all CNN models were over 0.90, and the area under the receiver operating characteristic curve reached 0.995 in the ResNet152 model. The pixel-wise discriminative features and the heat map of the prediction layer in the VGG16 model both revealed it focused on the largest gradient difference of topographic maps, which was corresponding to the diagnostic clues of ophthalmologists. The subclinical keratoconus was positively predicted with our model and also correlated with topographic indexes. Conclusions: The DL models had fair accuracy for keratoconus screening based on corneal topographic images. The visualization mentioned in the current study revealed that the model focused on the appropriate region for diagnosis and rendered clinical explainability of deep learning more acceptable. Translational Relevance: These high accuracy CNN models can aid ophthalmologists in keratoconus screening with color-coded corneal topography maps.

Therapeutic penetrating keratoplasty for microbial keratitis in Taiwan from 2001 to 2014.

PURPOSE: To study the surgical outcome of therapeutic penetrating keratoplasty (TPK) for medically uncontrolled microbial keratitis at a tertiary hospital in Taiwan over a 14-year period. METHODS: This is a retrospective case series study. Patients who underwent TPK at National Taiwan University Hospital in 2001-2014 were included. Patients were divided into 3 diagnostic groups: bacterial keratitis, fungal keratitis, and acanthamoeba keratitis. Each of the following criteria was evaluated: graft clarity at 1 month and 1 year postoperatively, cure of the disease, and anatomical success rate. RESULTS: A total of 107 TPKs were included. TPK eradicated the infection in 57/62 (91.9%) of bacterial keratitis, 33/41 (80.5%) of fungal keratitis, and 9/10 (90.0%) of acanthamoeba keratitis. 22/57 grafts (38.6%) of bacterial keratitis, 22/38 grafts (57.9%) of fungal keratitis, and 5/10 grafts (50.0%) of acanthamoeba keratitis remained clear at 1 year postoperatively. The 1-year graft survival rate did not significantly differ among these 3 groups. The leading causes of graft failure were late endothelial decompensation and graft reinfection. A higher percentage of graft clarity was achieved in grafts <8.5 mm in diameter than in larger grafts (55.4% vs. 31.4%, P = 0.027). Of the 14 patients with endophthalmitis at the time of TPK, the infection was successfully treated in 13 patients except 1 patient required evisceration. CONCLUSION: TPK is valuable in the management of medically uncontrolled microbial keratitis, although the 1-year graft survival rate is unsatisfactory. TPK combined with intravitreal antibiotics and vitrectomy is also a beneficial treatment strategy for patients with endophthalmitis secondary to microbial keratitis.

Knockdown of IQGAP-1 Enhances Tight Junctions and Prevents P. aeruginosa Invasion of Human Corneal Epithelial Cells.

PURPOSE: To determine the role of IQ-domain GTPase-activating protein1 (IQGAP-1) in tight junctions of human corneal epithelial cells (HCECs) and its effect against P. aeruginosa (PAK) invasion. MATERIAL AND METHODS: Primary human corneal epithelial cells (HCECs), immortalized HCECs, and IQGAP-1 RNA knockdown HCECs (siHCECs) were used. Confocal microscopy, transepithelial electrical resistance (TER), trypan blue exclusion assay and gentamicin invasion assay were done. RESULTS: In primary and immortalized HCECs, IQGAP-1 co-localized with zonular occludin-1 (ZO-1) and actin. Enhanced actin and ZO-1 aggregation were seen in siHCECs. IQGAP-1 knockdown significantly increased TER of immortalized HCECs (P < .0001). Cell viability after PAK infection increased for siHCECs for up to 4 h after infection. PAK intracellular invasion was significantly lowered by 50% in siHCECs at 1 h post-infection. CONCLUSION: IQGAP-1 knockdown increased the strength and integrity of tight junctions and may provide an early protective effect against P. aeruginosa invasion.

Clinical features and outcomes of Acanthamoeba keratitis in a tertiary hospital over 20- year period.

PURPOSE: To analyze the outcomes of Acanthamoeba keratitis (AK) in terms of different clinical presentations in a tertiary hospital in Taiwan over a 20- year period. METHODS: This is a retrospective case series. Patients with AK diagnosed at the National Taiwan University Hospital between January 1996 and December 2015 were identified. A diagnosis of AK was made on the basis of positive Acanthamoeba smear/cultures or pathological identification of Acanthamoeba cysts on keratoplasty specimens. Patient demographics, clinical presentations, treatment courses, and final visual outcomes were collected and analyzed. Visual acuity, postoperative complications and graft survivals were measured as outcomes. RESULTS: Of the 62 patients with AK in our study, 64.5% were medically treated while 35.5% underwent surgical treatment. In those with ring infiltrate, 52.4% patients could be successfully treated with medications. In eyes receiving penetrating keratoplasty, postoperative complications were more common in therapeutic penetrating keratoplasty (TPK) than those in optical penetrating keratoplasty (OPK) group (82.4% versus 40%, p = 0.04). The graft size was larger in TPK than that in OPK group (8.56 versus 7.83 mm, p = 0.002). Furthermore, post-operative glaucoma, which was the major complication, was found to be associated with larger graft size (p = 0.02) and dilated pupil/iris atrophy (p = 0.01). CONCLUSION: Even in advanced cases with ring infiltrate, eradication of infection with anti-amoebic drugs is possible. In those requiring keratoplasty, the surgical timing should be made meticulously considering graft size and signs of dilated pupil/iris atrophy in terms of post-operative glaucoma.

Analysis of P. aeruginosa disinfectant sensitivity and microbial adhesions to worn cosmetic contact lenses.

PURPOSE: To compare the sensitivity of two genotypes of P. aeruginosa to various disinfectant solutions and analyze the attached bacteria on worn cosmetic contact lenses (cosCLs). METHODS: In this prospective study, healthy volunteers wore etafilcon (brown), nelfilcon (gray), or hilafilcon (black) cosCLs and microbial adhesion analysis was performed. A rub-off test determined pigment dislodgement. Disinfectant sensitivity to Optifree Replenish (Alcon), Optifree Pure Moist (Alcon), Renu Fresh (Bausch & Lomb), and AoSept Plus (Ciba Vision) was tested at various disinfection times and compared between various genotypes and Type III secretion (T3S) system mutants. RESULTS: Of the 1152 cosCLs collected, 364 were culture positive (32%). The highest rate of culture-positive lens was hilafilcon (chi square, P = 0.0001). Hilafilcon also had a significantly greater number of isolates than etafilcon (P < 0.0001). Hilafilcon was the only lens to fail the rub-off test. Cytotoxic strains were significant more resistant to Renu Fresh than were invasive strains, even at 100% of recommended disinfection time (P = 0.0005). Of the tested disinfectants, Renu Fresh was significantly less effective in killing both genotypes of P. aeruginosa compared to AoSept Plus at all time points (25%, 50%, 75%, and 100% recommended disinfection time, P = 0.0001, 0.0001, 0.0005, and 0.0005, respectively). When the T3S system was dysfunctional, mutant strains were all susceptible to disinfectants (P = 0.0001 for both invasive and cytotoxic strains). CONCLUSION: Pseudomonas species is commonly found on cosCLs of asymptomatic individuals. Wearers of cosCLs that dislodge pigments may be predisposed to microbial contamination. Cytotoxic strains are more resistant to disinfectant solutions, especially to Renu Fresh. P. aeruginosa disinfectant resistance requires a functional T3S system.