Complement Cascade 8 - Alpha and Calpain-2 in Extracellular Vesicles of Human Vitreous as Biomarkers of Infectious Endophthalmitis.

Purpose: Extracellular vesicles (EVs) are messenger pigeons of the cells that communicate about cellular microenvironment. In this study, we evaluated the expression of C8α and calpain-2 in EVs from vitreous of patients with bacterial endophthalmitis to assess its utility as a diagnostic marker. Methods: EVs were isolated from vitreous of patients with bacterial endophthalmitis (culture positive and culture negative) and noninfectious control by exosome isolation reagent and characterized, and the levels of C8α and calpain-2 was assessed by enzyme-linked immunosorbent assay in isolated EVs and direct vitreous. The receiver operating characteristic curve was generated to assess the diagnostic performance. Results: Scanning electron microscopy (SEM) and dynamic light scattering (DLS) confirmed the presence of EVs having a diameter (nm) of 275.2 ± 93, 92 ± 22, and 77.28 ± 12 in culture-positive (CP), culture-negative (CN), and control respectively. The expression level (ng/mL) of C8α in the EVs obtained from CP was 144 ± 22 and CN was 31.2 ± 9.8, which was significantly higher (P < 0.01) than control 3.7 ± 2.4. Interestingly, C8α is not expressed directly in the vitreous of CN and controls. Calpain-2 was significantly downregulated (P ≤ 0.0001) in CP (0.94 ± 0.16) and CN (0.70 ± 0.14) than control. The sensitivity and specificity of 1 for C8α and calpain-2 in the EVs implied that its diagnostic accuracy was significant. Conclusions: This study showed that the EV proteins C8α and calpain-2 could be suitable diagnostic markers for endophthalmitis. However, the presence of C8α in the EVs of CN samples but not in direct vitreous promises EVs as the future of diagnostics. Translational Relevance: Expression levels of EV-calpain-2 and EV-C8α could diagnose CN bacterial endophthalmitis.

Macular Pigment Assessment in Indian Population Using Degree of Polarization Threshold: Impact of Diet on Macular Pigment Density.

Purpose: To determine macular pigment (MP) density scores in healthy Indians and examine correlations with demographic and lifestyle variables. Methods: We observed 484 Indians without an ocular pathology. Body mass index (BMI) and self-reported lifestyle factors (sunglasses usage, physical activity, and smoking) were noted. MP density was assessed as the threshold of perception of the shadow of their macular pigments on their retina using a new MP assessment tool (MP-eye). Lutein and zeaxanthin intake was assessed using a prevalidated questionnaire regionally designed for the Indian diet. Clusters of participants were created for statistical analysis based on MP-eye scores secondarily to detect any relevant effects in very low, low, medium, and high ranges of MPs. Results: Data analyzed included 235 males and 249 females with mean age of 36.1 ± 12.9 years (range, 14-72). The median MP-eye score was 6 (range, 0-10, with 10 being high). Most were non-smokers (413, 85.3%) and did not use sunglasses (438, 90.5%), and 314 (64.9%) had low physical activity. Diabetes was present in 62 participants (12.8%) and hypertension in 53 (10.9%). Advancing age (r = -0.209; P < 0.000) and BMI (r = -0.094; P = 0.038) had weak negative correlation with MP-eye scores. Hypertension was less prevalent (7/88) in the cluster with the highest median MP-eye score (P = 0.033). Dietary intake of MPs and other lifestyle factors did not correlate significantly with MP-eye score overall or when analyzed in clusters. Conclusions: MP-eye scores of an Indian population were normally distributed. Higher age, high BMI, and presence of hypertension were weakly associated with lower MP-eye scores. The impact of diet on MPs requires further evaluation. Translational Relevance: This normative regional database enables risk stratification of macular degeneration.

Focused analysis of RNFL decay in glaucomatous eyes using circular statistics on high-resolution OCT data.

We generated Optical Coherence Tomography (OCT) data of much higher resolution than usual on retinal nerve fiber layer (RNFL) thickness of a given eye. These consist of measurements made at hundreds of angular-points defined on a circular coordinate system. Traditional analysis of OCT RNFL data does not utilize insightful characteristics such as its circularity and granularity for common downstream applications. To address this, we present a new circular statistical framework that defines an Angular Decay function and thereby provides a directionally precise representation of an eye with attention to patterns of focused RNFL loss. By applying to a clinical cohort of Asian Indian eyes, the generated circular data were modeled with a finite mixture of von Mises distributions, which led to an unsupervised identification in different age-groups of recurrent clusters of glaucomatous eyes with distinct directional signatures of RNFL decay. New indices of global and local RNFL loss were computed for comparing the structural differences between these glaucoma clusters across the age-groups and improving classification. Further, we built a catalog of directionally precise statistical distributions of RNFL thickness for the said population of normal eyes as stratified by their age and optic disc size.

Circular functional analysis of OCT data for precise identification of structural phenotypes in the eye.

Progressive optic neuropathies such as glaucoma are major causes of blindness globally. Multiple sources of subjectivity and analytical challenges are often encountered by clinicians in the process of early diagnosis and clinical management of these diseases. In glaucoma, the structural damage is often characterized by neuroretinal rim (NRR) thinning of the optic nerve head, and other clinical parameters. Baseline structural heterogeneity in the eyes can play a key role in the progression of optic neuropathies, and present challenges to clinical decision-making. We generated a dataset of Optical Coherence Tomography (OCT) based high-resolution circular measurements on NRR phenotypes, along with other clinical covariates, of 3973 healthy eyes as part of an established clinical cohort of Asian Indian participants. We introduced CIFU, a new computational pipeline for CIrcular FUnctional data modeling and analysis. We demonstrated CIFU by unsupervised circular functional clustering of the OCT NRR data, followed by meta-clustering to characterize the clusters using clinical covariates, and presented a circular visualization of the results. Upon stratification by age, we identified a healthy NRR phenotype cluster in the age group 40-49 years with predictive potential for glaucoma. Our dataset also addresses the disparity of representation of this particular population in normative OCT databases.

Analysis of yield of retinal imaging in a rural diabetes eye care model.

PURPOSE: The aim of this study is to analyze the yield of retinal images obtained in a rural diabetes eye care model. METHODS: An analysis of a sample of nonmydriatic fundus photography (NMFP) of posterior segment ophthalmic images, obtained by an indigenous equipment (3 nethra-Forus Royal), was done in a district-wide rural diabetic retinopathy (DR) screening program; a trained optometrist did the initial image grading. DR and diabetic macular edema (DME) were classified based on international DR and DME severity scale. The agreement between the optometrist and retina specialist was very good (κ = 0.932; standard error = 0.030; 95% confidence interval = 0.874-0.991). RESULTS: Posterior segment images of 2000 eyes of 1000 people with diabetes mellitus (DM) were graded. The mean age of the participants was 55.7 ± 11.5 standard deviation years. Nearly 42% of the screened participants (n = 420/1000) needed referral. The most common referable posterior segment abnormality was DR (8.2%). The proportion of people with any form of DR was seen in 110/1225 eyes, and sight-threatening DR was seen in 35/1225 eyes. About 62% of posterior segment images were gradable. The reasons for ungradable posterior segment images (34%) were small pupil, unfocused/partially available field of images, and cataract. CONCLUSION: A NMFP model was able to detect referable posterior segment abnormalities in a rural diabetes eye care program. Reasons found for ungradability of images in the present study can be addressed while designing future DR screening programs in the rural areas.

Multidrug-Resistant Pseudomonas aeruginosa Keratitis: Risk Factors, Clinical Characteristics, and Outcomes.

PURPOSE: To identify risk factors for multidrug-resistant Pseudomonas aeruginosa (MDR-PA) keratitis and to report clinical characteristics and outcomes of this infection. DESIGN: Retrospective, case-control study. PARTICIPANTS: Ninety episodes of P. aeruginosa keratitis from a tertiary care eye institution from 2007 through 2014 were identified. Episodes of MDR-PA keratitis were taken as cases. Episodes of drug-sensitive P. aeruginosa keratitis during the same period were taken as controls. METHODS: Chart review of cases and controls. Multidrug resistance was defined as resistance to 3 or more classes of antimicrobial drugs. Logistic regression analysis was used to determine association of baseline characteristics with multidrug resistance. Clinical characteristics and risk factors in each group also were compared. MAIN OUTCOME MEASURES: Risk factors for multidrug resistance, including age, gender, eye, duration of symptoms, topical steroid or lubricant ointment use, trauma, prior ocular surgery, presence of a compromised ocular surface, and bandage contact lens use. RESULTS: Twenty-three episodes of MDR-PA keratitis (cases) and 67 episodes of drug-sensitive P. aeruginosa keratitis (controls) were identified. In the multivariate analysis, lubricant ointment use, presence of a compromised ocular surface, and bandage contact lens use were associated with MDR-PA keratitis. Antimicrobial resistance in the MDR-PA keratitis isolates was least for colistin and imipenem (56.52% each). Corneal perforation was more common in cases (52.17% [12 of 23]) than in controls (11.94% [8 of 67]; P = 0.0001). Cyanoacrylate glue application was required in 47.82% of cases (11 of 23), compared with 22.38% of controls (15 of 67; P = 0.031). Keratoplasty was more commonly required in cases (47.82% [11 of 23]) than in controls (20.89% [14 of 67]; P = 0.017). CONCLUSIONS: Lubricant ointment use, a compromised ocular surface, and bandage contact lens use are associated with MDR-PA keratitis. Preservative-free lubricant ointment may act as a source or reservoir of infection. Outcomes are worse in MDR-PA keratitis compared with drug-sensitive P. aeruginosa keratitis.

Re-appraisal of topical 1% voriconazole and 5% natamycin in the treatment of fungal keratitis in a randomised trial.

PURPOSE: To compare the efficacy of topical 1% voriconazole vs 5% natamycin for the treatment of fungal keratitis. METHODS: In a prospective, double-masked, randomised, controlled, registered clinical trial, 118 patients with fungal keratitis were treated using identical dosage schedule with either voriconazole (58) or natamycin (60) as inpatients for 7 days and followed up weekly. The outcome measures were percentage of patients with healed or resolving ulcer and final visual acuity at last follow-up (primary) and on day 7 (secondary) in each group. RESULTS: More patients (p=0.005) on natamycin (50/56, 89.2%) had healed or resolving ulcer compared with voriconazole (34/51, 66.6%) at last follow-up. The improvement in vision was marginally greater in patients in the natamycin group compared with the voriconazole group at day 7 (p=0.04) and significantly greater at final visit (p=0.01). In univariate analysis, drug, age and mean size of corneal infiltrate and epithelial defect had a significant effect on the final visual outcome. In multivariate analysis, the effect of drug (voriconazole vs natamycin, adjusted coefficient 0.27 (-0.04 to 0.57), p=0.09) was marginal while the effect of age and epithelial defect was significant (p<0.001 for both). In the group treated with natamycin, the final visual acuity was significantly better (p=0.005, Wilcoxon signed-rank test) in patients with Fusarium keratitis but not with Aspergillus keratitis (p=0.714, paired t test). CONCLUSIONS: When compared with voriconazole, natamycin was more effective in the treatment of fungal keratitis, especially Fusarium keratitis. TRIAL REGISTRATION NUMBER: Clinical Trial Registry India (2010/091/003041).

Vitreous haemorrhage in massive hemorrhagic polypoidal choroidal vasculopathy: clinical characteristics and surgical outcomes: Vitreous hemorrhage in PCV.

BACKGROUND: To report the outcomes of vitreous hemorrhage (VH) associated with hemorrhagic polypoidal choroidal vasculopathy (PCV). METHODS: A retrospective study of 28 eyes of 27 consecutive patients of hemorrhagic PCV with VH, which were managed surgically between January 2003 and December 2011, was performed. All patients underwent pars plana vitrectomy for VH associated with PCV. The main outcome measure was best-corrected visual acuity (BCVA) at baseline, at 1, 3 and 6 months post operatively and at last follow up. RESULTS: The visual acuity measured on early treatment diabetic retinopathy study (ETDRS) chart improved in 16 eyes (57.1 %) by two or more lines, remained unchanged in nine eyes (32.1 %) and decreased in three (10.7 %) after surgery when compared to baseline VA. The mean baseline VA was 2.69 ± 0.57 logMAR units (<20/2000) which improved to 1.65 ± 0.93 logMAR units (20/800) at 1 month post operative visit and was sustained at 1.72 ± 1.12 (20/800) with an improvement of 0.96 logMAR units (p < 0.001, 95 % CI 0.54-1.37). The average postoperative follow up was for 14.2 months (range 1-84). The complications noted in postoperative follow up were cataract (n = 10), macular scaring (n = 9), organised dehemoglobinised blood (n = 7), retinal tear or detachment (n = 5), recurrent VH (n = 3) and choroidal detachment (n = 1). CONCLUSION: Majority of patients with loss of vision due to VH secondary to hemorrhagic PCV have sustained improvement in visual acuity following surgery.

Unilateral partial limbal stem cell deficiency: contralateral versus ipsilateral autologous cultivated limbal epithelial transplantation.

PURPOSE: To report the outcomes of autologous cultivated limbal epithelial transplantation using the healthy part of the affected eye or the fellow eye as a source of limbal stem cells in patients with unilateral, partial limbal stem cell deficiency (LSCD). DESIGN: Retrospective, nonrandomized, interventional case series. METHODS: setting: L. V. Prasad Eye Institute, Hyderabad, India. study population: Patients with unilateral, partial LSCD who underwent autologous cultivated limbal epithelial transplantation between 2001 and 2011. intervention: The limbal biopsy was taken either from the healthy part of the limbus of the same eye (ipsilateral group) or from the healthy fellow eye (contralateral group). Cells were cultivated using a xeno-free explant culture technique, and cultivated cells were transplanted onto the affected surface. primary outcome measure: Success of cultivated limbal epithelial transplantation, defined as a completely epithelialized, avascular, and clinically stable corneal surface. RESULTS: Seventy eyes of 70 patients were studied. The mean follow up was 17.5 ± 7 months. In 34 eyes the limbal biopsy was taken from the ipsilateral eye and in the remaining 36 eyes from the contralateral eye. Clinical success was achieved in 70.59% of eyes in the ipsilateral group and 75% of eyes in the contralateral group (P = .79). Limbal transplant survival rates at the final follow-up visit were 65.1% ± 0.09% in the ipsilateral group and 53.6% ± 0.12% in the contralateral group (P = .74). CONCLUSIONS: Ocular surface restoration in partial LSCD is possible with cell-based therapy. Outcomes are similar irrespective of whether the limbal biopsy is taken from the healthy part of the ipsilateral eye or the contralateral eye.

A 3½-Year Study of Bacterial Keratitis From Odisha, India.

PURPOSE: To report the clinical and microbiological characteristics and treatment outcome of bacterial keratitis at a tertiary eye care center in eastern India. DESIGN: Retrospective analysis of medical and microbiology records. METHODS: All patients had undergone complete clinical and microbiological evaluation for microbial keratitis. Patients with laboratory-proven bacterial keratitis were included in the study. RESULTS: Between July 2006 and December 2010, 1417 microbial keratitis was clinically diagnosed in the patients. Whereas no organisms were found in 27.8% (394/1417) of cases, 21.4% (303/1417) were bacterial. From 303 patients, 347 bacterial isolates were cultured, 260 (74.9%) of which were gram-positive, 67 were gram-negative, and 20 were acid-fast. Streptococcus pneumoniae was the predominant isolate (86/347 [24.7%]), followed by Staphylococcus species [64/347 (18.4%)]. Pseudomonas aeruginosa (29/347 [8.3%]) was the most common gram-negative bacterial isolate. High level of susceptibility to cefazolin (96.2%) and vancomycin (96.5%) was found in gram-positive cocci, whereas susceptibility of Pseudomonas species to gatifloxacin was 95.1%. Fifty-three patients (17.5%) required tissue adhesive, and 47 (15.5%) needed penetrating keratoplasty. Healed corneal scar was achieved in 188 patients (62%), whereas 34 (11.2%) were lost to follow-up. Large stromal infiltrate size, older age group, and poor presenting visual acuity were significant factors that adversely affected final outcome (P < 0.05). CONCLUSIONS: Proportion of bacterial keratitis was low compared with other studies from India. Gram-positive bacteria were a common cause of bacterial keratitis with high susceptibility to cefazolin and vancomycin. Gram-negative bacteria were sensitive to gatifloxacin with overall good treatment outcome.

Mitomycin C in dacryocystorhinostomy: the search for the right concentration and duration--a fundamental study on human nasal mucosa fibroblasts.

PURPOSE: To establish primary cultures of human nasal mucosal fibroblasts (HNMFs) and to test the effect of varying concentrations of mitomycin C (MMC) and treatment durations on cellular proliferation and viability of the fibroblasts. DESIGN: Laboratory investigation. METHODS: Nasal mucosa harvested from patients undergoing a dacryocystorhinostomy was used to establish primary cultures by explant culture method. Cells were expanded and frozen at every passage, and passage 3 cells were used for further experiments. The cells were then treated with different concentrations of mitomycin C (0.1-0.5 mg/ml) for different time periods (3, 5, and 10 minutes). Cell viability was checked by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cellular proliferation index was determined with bromodeoxyuridine immunostaining. Apoptotic index was measured using annexin A5 affinity assay, propidium iodide staining, and 4',6-diamidino-2-phenylindole counterstaining. The actin cytoskeletons of fibroblasts were studied using phalloidin staining. RESULTS: The doubling time of cultured HNMFs is approximately 24 hours. Similarly, 0.4 mg/ml beyond 5 minutes and 0.5 mg/ml concentration at all time points were lethal and caused extensive cell death when compared with controls. A concentration of 0.2 mg/ml for 3 minutes of exposure prevented cell proliferation of HNMF cells by inducing cell cycle arrest, without causing extensive apoptosis. CONCLUSIONS: The minimum effective concentration appears to be 0.2 mg/ml for 3 minutes. This in vitro study could be the starting point for further clinical and histopathologic studies to validate its clinical usefulness.

Anatomic and visual outcomes of descemetopexy in post-cataract surgery descemet's membrane detachment.

OBJECTIVE: To study the anatomic and visual outcomes of descemetopexy in Descemet's membrane detachment (DMD) after cataract surgery. DESIGN: Retrospective case series. PARTICIPANTS: Clinical notes of 60 patients who underwent DMD after cataract surgery between 2007 and 2011. METHODS: Descemetopexy was performed with air or 14% isoexpansile perfluoropropane (C3F8). MAIN OUTCOME MEASURES: Anatomical (reattachment rates) and functional results (best-corrected visual acuity) were studied. Secondary outcome measures were assessment of surgical complications and association of various factors with final visual outcome. RESULTS: The mean age of the patients was 64.3 ± 8.3 years, and the male:female ratio was 21:39. At 1 month, the mean logarithm of the minimum angle of resolution (logMAR) interval visual acuity (IVA) improved from 1.27 ± 0.8 to 0.42 ± 0.49 (P < 0.001). Five patients (8.3%) obtained 20/20 vision, and 37 of 60 patients (61.6%) achieved IVA of ≥ 20/40. Ninety-five percent (57/60) of patients had successful reattachment of the Descemet's membrane (DM) after the intervention. Multiple linear regression analysis showed that patients with a cataract score of 5 (estimate = 0.38; P=0.014), with a cataract score of 4 with compromised visibility due to a corneal opacity (estimate = 0.45; P=0.039), and prolonged duration between cataract surgery and descemetopexy (estimate = 0.012; P=0.007) were associated with a significantly poorer final visual outcome. No association of final visual outcome was observed with age; sex; eye treated; cataract scores 2, 3, and 4; preoperative visual acuity; and involvement of the visual axis (P > 0.5). The eyes in which air was used for descemetopexy (estimate = -0.2; P=0.009) had statistically significantly better final visual outcomes. Three patients (5%) had treatment failures and required subsequent endothelial transplantation. Pupillary block was observed in the early postoperative period in 7 patients (11.66%) in whom C3F8 had been used and was not seen with air (P=0.02). CONCLUSIONS: This study suggests that DMD after cataract surgery can be treated effectively and good visual outcomes can be expected if the patient is treated in time with anterior chamber injection of gas. Air has advantages of better efficacy than C3F8 without the risk of pupillary block and thus should be preferred.

Anterior segment optical coherence tomography features of acute corneal hydrops.

PURPOSE: To describe the clinical significance of in vivo corneal findings in eyes with acute corneal hydrops. METHODS: Medical records and anterior segment optical coherence tomography (ASOCT) images of 24 patients with keratoconus and unilateral acute corneal hydrops were reviewed. Clinical findings and position, integrity, and orientation of the Descemet membrane (DM) on ASOCT were noted. Size of the DM break, depth of the DM detachment, and corneal thickness were measured by ASOCT and correlated with the duration of corneal edema, assessed clinically. Outcomes of intracameral perfluoropropane (C(3)F(8)) gas in 13 eyes and histopathological findings after penetrating keratoplasty in 2 eyes were also correlated with serial ASOCT findings. RESULTS: Duration of corneal edema was 9.3 ± 4.4 weeks. On ASOCT, DM showed 3 patterns: detachment with break and rolled ends (n = 13), detachment with break and flat ends (n = 10), and detachment with no break (n = 1). Initial corneal thickness was 1.3 ± 0.25 mm, size of the DM break was 1.1 ± 0.8 mm, and depth of the DM detachment was 0.9 ± 0.6 mm. On multiple regression analysis, duration of corneal edema showed significant positive association with the depth of DM detachment (P = 0.0002) and size of DM break (P = 0.002) and negative association with intracameral C3F8 (P < 0.0001). Correlation of ASOCT with histopathological findings revealed 2 stages of resolution of hydrops: reattachment of DM and endothelial migration. CONCLUSIONS: Eyes with deeper DM detachments and larger DM breaks required more time for resolution of corneal edema despite intracameral C(3)F(8). This association needs to be validated by a prospective study.