Three cases of recalcitrant Paecilomyces keratitis in Southern California within a short period.

BACKGROUND: The aim of this report is to describe the risk factors, clinical course, and characteristics of three cases of Paecilomyces keratitis presenting concurrently within three months in the same location. We used in vivo confocal microscopy and histopathology to corroborate our clinical findings. OBSERVATIONS: Three eyes of three elderly patients with culture-proven Paecilomyces keratitis were included in this series. These patients resided within a 15-mile radius and presented to a tertiary care eye institute in Southern California between February and April 2022. All three eyes experienced a prolonged, recalcitrant course with recurrence of keratitis in donor corneal tissue despite antifungal therapy and multiple therapeutic penetrating keratoplasties. In vivo confocal microscopy, histopathology, and microbiologic findings corroborated the diagnosis of fungal keratitis with Paecilomyces. With surgical intervention and extensive medical therapy, all three cases resolved after the addition of oral Posaconazole. CONCLUSIONS: Paecilomyces is a rare cause of infectious keratitis. Herein we report three similar cases in elderly patients. All had prolonged, recalcitrant infections that required multiple treatment modalities. Our cases, which were supported by in vivo confocal microscopy and histopathology, highlight the importance of timely and aggressive therapy to prevent recurrence.

Risk Factors for Meibomian Gland Disease Assessed by Meibography.

Purpose: To elucidate risk factors for meibomian gland disease (MGD) and understand associated changes in meibography and in relation to ocular surface disease. Patients and Methods: As part of the standard workup for ocular surface disease at a tertiary academic center, 203 patients received an ocular history and lifestyle questionnaire. The questionnaire included detailed inquiries about ocular health and lifestyle, including makeup use, cosmetic eyelid procedures, screen time, and contact lens habits. Subjects also took the standardized patient evaluation of eye dryness (SPEED) II questionnaire. Meibomian gland (MG) dropout and structural changes were evaluated on meibography and scored by three independent graders using meiboscores. Statistical analysis was conducted to identify significant risk factors associated with MG loss. Results: This retrospective, cross-sectional study included 189 patients (378 eyes) with high-quality images for grading, and the average age was 67 years (77% female). Patients older than 45 years had significantly more dropout than younger patients (p < 0.01). Self-reported eye makeup use did not significantly impact MG loss. Patients with a history of blepharoplasty trended toward higher meiboscores, but the difference was not statistically significant. Self-reported screen time did not affect meiboscores. Contact lens use over 20 years was associated with significant MG loss (p < 0.05). SPEED II scores had no relationship to meiboscores (p = 0.75). Conclusion: Older age is a significant risk factor for MG loss. Any contact lens use over 20 years also impacted MG dropout. Highlighting the incongruence of symptoms to signs, SPEED II scores showed no relationship to the structural integrity of MGs.

Quantifying the Size of the Pterygium Head by Evaluating the Fibrous Area or the Vascularized Area.

PURPOSE: Pterygium is characterized by the development of wing-shaped fibrovascular tissue from the bulbar conjunctiva across the limbus onto the cornea. The purpose of this study was to quantify the discrepancy of the pterygium size between the fibrous area and the vascularized area. METHODS: Color photographs of 146 eyes with pterygia obtained with a single-lens reflex camera system were analyzed by 2 independent graders for width (vertical dimension), length (horizontal dimension), and surface area of the pterygium head using 2 methods: the fibrous method, which measures any fibrovascular tissue crossing the corneal limbus, and the vascular method, which measures only tissue with discernible active blood flow, without any avascular tissue or opacity. Statistics of intraclass correlation coefficients for intergrader and intragrader reproducibility were calculated, and the paired t test between methods was used. RESULTS: Both intergrader and intragrader intraclass correlation coefficients for both methods were above 0.85. The vascular measurement was significantly larger in width (vertical dimension, fibrous 3.97 ± 1.02 mm vs. vascular 4.49 ± 1.33 mm, P = 0.01), whereas the fibrous measurement was significantly larger in length (horizontal dimension, fibrous 2.41 ± 1.16 mm vs. vascular 2.23 ± 1.05 mm, P = 0.04). No significant difference was found in the surface area (fibrous 7.27 ± 4.65 mm vs. vascular 7.40 ± 5.13 mm, P = 0.67). CONCLUSIONS: With both methods, reproducibility of the size of the pterygium head was high. Significant differences were shown between the two methods in quantification of the width and length but not in the surface area. Such methods can be used to standardize the evaluation of pterygia in clinical research and clinical trials.

Discrepancies in Endothelial Cell Density Values of Human Donor Corneas Resulting From Comparison Between Specular Microscopes and Endothelial Analysis Methods.

PURPOSE: The methods for specular microscopy evaluation across eye banks differ, which may result in variability in endothelial cell density (ECD) values that influence the surgeon's decision about donor tissue. A comparison of instruments and analysis methods is conducted in this study. METHODS: Specular images were captured from 97 donor corneas using both HAI and Konan specular microscopes. A single best quality image of each cornea from each instrument was graded using the respective inherent software and analysis method (HAI: variable frame method; Konan: center method). All raw specular images were standardized for dimensions and regraded in the CellChek system in a blinded fashion. The grading variances and paired t test were performed between instruments in both inherent and standardized analyses. Correlation and Bland-Altman analyses between instruments were also performed. RESULTS: Using the software inherent within HAI and Konan, the mean ECD readings for the 97 corneas were 2764 ± 583 and 2605 ± 517 cell/mm (P = 0.045), respectively, with a variance of 8.05% (range 0.26%-27.2%). HAI resulted in a higher ECD value in 79 corneas (81.4%). In CellChek software analysis, the mean ECD readings did not differ (2609 ± 514 and 2496 ± 507 cells/mm, respectively, P = 0.127), with a variance of 5.6% (range 0.24%-19.8%). CONCLUSIONS: There is a slight statistically significant mean difference between the ECD values obtained from the 2 specular microscopes, which is negated by standardization to a single analysis method. Eye banks and surgeons should use caution in making decisions based only on very small differences in ECD between otherwise equivalent corneal donor tissues.

Role of in vivo confocal microscopy in the diagnosis of infectious keratitis.

PURPOSE: To investigate the utility of in vivo confocal microscopy (IVCM) in the diagnosis of infectious keratitis (IK). METHODS: Retrospective chart review of 46 patients with a final diagnosis of IK were included in the study. All patients received IVCM corneal imaging using the Heidelberg Retinal Tomography III system. All available scans were randomized and analyzed in a masked fashion. Sensitivity and specificity of IVCM in diagnosing bacterial keratitis (BK), Acanthamoeba keratitis (AK), fungal keratitis (FK), and HSV viral keratitis (VK) were assessed. RESULTS: The pooled sensitivity and specificity of IVCM in identifying atypical IK (AK and FK cases combined) were 85.3% (95% CI 68.2-94.5%) and 100% (95% CI 74.7-100%), respectively. The sensitivity and specificity of IVCM in identifying BK were 66.7% (95% CI 35.4-88.7%) and 89.2% (95% CI 73.4-96.5%), respectively. The sensitivity and specificity of IVCM in identifying VK were 100% (95% CI 46.3-100%) and 93.2% (95% CI 80.3-98.2%). Additionally, IVCM was able to make the correct diagnosis in 8 out of the 11 atypical keratitis cases misdiagnosed clinically. In the AK subgroup, IVCM was more accurate than clinical assessment (16 vs. 11). In the FK subgroup, IVCM were as accurate as clinical assessment, but did correct one misdiagnosed cases by identfying fungal hyphae. CONCLUSION: IVCM is an non-invasive imaging modality that can rapidly and accurately diagnose IK even for experienced corneal specialists. In complex cases of polymicrobial infection, IVCM may guide the correct clinical diagnosis and initiation of the appropriate treatment.

Representation of Central Endothelial Cell Density by Analysis of Single Best Specular Microscopy Image Regardless of Cell Size Variance.

PURPOSE: The purpose of this study is to evaluate whether a single best image can represent central endothelial cell density (ECD) in corneas of differing cell size coefficient of variance (CV). METHODS: Four hundred one healthy eyes but with variant CV values were enrolled. For each eye, three nonoverlapping central cornea endothelium images were obtained with Konan NSP-9900 specular microscope. ECD and CV were evaluated by two independent graders using the well-established Center method. Only corneas with high image quality rating (IQR) and ECD >800 cell/mm2 by both graders were included in the study. The study sample was stratified into five CV levels (CV ≤ 35; ≥36; ≥38; ≥40; and ≥45). In each CV level, the ECD agreement, ECD variance, and the correlation between the ECD variation and CV values were analyzed. In addition, the ECD intragrader reproducibility and interframe differences were also analyzed for all levels except CV ≤ 35. RESULTS: The study sample includes a total of 278 eyes. High ECD agreement for the two independent graders (intraclass correlation coefficient [ICC] > 0.99), high ECD intragrader reproducibility (ICC > 0.95), low ECD variance (2.0% ± 1.6%, overall), no correlation between the ECD variation and the CV value (P > 0.05), and no significant ECD difference among frames (P > 0.05) was found in any studied CV levels. CONCLUSIONS: CV does not appear to be associated with ECD variance in the central cornea. TRANSLATIONAL RELEVANCE: This finding highlights that in healthy corneas but with high CV values, ECD can be reliably analyzed using one single image of best quality.

Correlation between Guttata Severity and Thickness of Descemet's Membrane and the Central Cornea.

Purpose: To characterize and correlate guttata severity, Descemet's membrane thickness (DMT), central cornea thickness (CCT) in corneas with guttae using specular microscopy and spectral-domain optical coherence tomography (SD-OCT) and test the Doheny Image Reading Center (DIRC) specular microscopy-based corneal guttata severity scale. Methods: Forty-nine eyes of 49 patients with guttata and 36 eyes of age-matched of 36 normal controls were enrolled in the study. Three images of the central cornea and four of the peripheral cornea (inferior, superior, nasal and temporal) of each eye were taken using the Konan NSP-9900 specular microscope. A volume scan of the central cornea cross-section was collected on each eye using the Heidelberg Spectralis SD-OCT. The density of endothelial guttata based on specular images was graded on a 0-4 scale, and the Descemet's membrane thickness (DMT) and central corneal thickness (CCT) were manually measured by two trained graders. Results: The DIRC corneal guttata severity scale showed good reproducibility of all corneal endothelial images (weighted Kappa = 0.87). Mean DMT was 16.1 ± 2.4 µm in controls and 25.5 ± 10.9 µm in corneas with guttata (P < 0.001). Mean CCT was 552 ± 26 µm in controls and 603 ± 55 µm in corneas with guttata (P < 0.001). Guttata severity was significantly correlated with both DMT (r = 0.743, P < 0.001) and CCT (r = 0.569, P < 0.001). Age was moderately correlated with DMT (r = 0.472, P = 0.003) and mildly correlated with guttata severity (r = 0.285, P = 0.031), but was not correlated with CCT (r = 0.058, P = 0.681). Guttatta grade 3 corneas displayed an increase in DMT and guttata grade 4 was associated with a significant increase in CCT. Conclusions: The DMT and CCT are increased in corneas with guttata. The higher density of guttae is correlated with increased thickness. Specular microscopy combined with SD-OCT can be used as a good approach to assess the severity of FECD.

Reduction of Acanthamoeba Cyst Density Associated With Treatment Detected by In Vivo Confocal Microscopy in Acanthamoeba Keratitis.

PURPOSE: Acanthamoeba keratitis (AK) is a severe vision-threatening ocular infection that is frequently a diagnostic challenge. Treatment course is lengthy and often not fully effective. Contact lens wear has been recognized as the prime risk factor for AK. In vivo confocal microscopy (IVCM) is a noninvasive imaging modality that allows direct visualization of potential causative pathogens in real time with an established utility in the diagnosis of AK. In this study, we aim to assess the utility of IVCM in monitoring disease progression in contact lens wearers with culture-confirmed keratitis. METHODS: Fourteen eyes from 11 patients with culture-confirmed AK were included in this retrospective study. IVCM was performed during the patient's initial visit and all follow-up visits. All available confocal sequences were reviewed and graded in a masked fashion. Density of Acanthamoeba cyst infiltration and changes in the cyst density as a percentage of baseline cyst density measured at each patient's initial visit were calculated. A univariate regression analysis was performed to assess the association between treatment and changes in cyst density per month of treatment. RESULTS: Acanthamoeba cysts were identified by IVCM in all of these culture-confirmed cases of keratitis. Mean cyst density in the central cornea at presentation was 99 ± 64.9 cells per square millimeter (range, 38-255/mm). Cyst density in our study population significantly decreased by approximately 5.3% with each month of antiamebic treatment (P = 0.001; R = 0.41). CONCLUSIONS: Reduction in Acanthamoeba cyst density with treatment can be monitored by IVCM, which in turn can be used clinically in prognostication and disease monitoring of AK.

Assessment of Corneal Changes Associated with Topical Antiglaucoma Therapy Using in vivo Confocal Microscopy.

PURPOSE: Ocular surface disease (OSD) is highly prevalent in eyes treated with chronic, topical antiglaucoma (A/G) therapy. The purpose of this study was to utilize in vivo confocal microscopy (IVCM) to evaluate the corneal morphology, including characteristics of corneal epithelial cells, presence of epithelial dendritic cells (DCs), and characteristics of subbasal nerve plexus, of eyes under topical A/G therapy versus normal eyes. METHODS: Central corneal images were prospectively captured from 30 eyes of 16 patients under topical A/G therapy (>6 months) and 20 normal control eyes, using IVCM (HRT 3 RCM, Heidelberg, Germany). Demographic data were collected, as well as information on the types and duration of A/G therapy. In addition, OSD index (OSDI) score, tear film breakup time, Schirmer 1 test results, density of epithelial wing cells (WCs) and basal cells (BCs), subbasal nerve features (density, tortuosity, and reflectivity), and presence of DCs were all assessed and recorded by trained Doheny Image Reading Center graders. RESULTS: IVCM findings of 30 glaucomatous eyes and 20 normal control eyes were analyzed. The mean OSDI score was 8.72 in controls and 32.06 in patients under A/G therapy (p = 0.002). Nerve fiber density, nerve fiber reflectivity, and BC density were all decreased in the A/G group (1,789.07 ± 785.70 µm/frame, 2.79 ± 0.83, 6,457.67 ± 692.55 cells/mm2, respectively) as compared to controls (2,815.981 ± 563.77 µm/frame, 3.52 ± 0.50, 7,854.13 ± 1,073.69 cells/mm2, respectively) (p < 0.05), whereas the decrease in WC density was statistically nonsignificant (p = 0.5). Nerve tortuosity and DC density were both significantly greater in the A/G eyes (3.00 ± 0.57, 71.24 ± 61.74 cells/mm2, respectively) compared to controls (2.10 ± 0.42, 34.08 ± 11.70 cells/mm2, respectively) (p < 0.05). Tear film breakup time and Schirmer 1 test results were significantly lower in the A/G group as compared to controls (p < 0.001). CONCLUSIONS: Using IVCM, our study identified significant microstructural alterations in the corneas of eyes treated with topical A/G therapy. In addition, our study also revealed that glaucoma patients treated with topical A/G therapy report significantly higher OSDI scores compared to controls. Thus, IVCM may be a useful tool in providing structural parameters to correlate with the functional OSDI assessments in the evaluation of ocular surface toxicity associated with topical A/G therapy.

Analysis of ocular inflammation in anterior chamber-involving uveitis using swept-source anterior segment OCT.

PURPOSE: To evaluate the utility of swept-source (SS) optical coherence tomography (OCT) to objectively analyze the degree of anterior chamber (AC) inflammation. METHODS: Thirty-eight eyes of 32 patients with uveitis and 20 control eyes were enrolled. SS OCT B-scans were obtained, and the number of cells in the B-scans was counted using two methods: (1) manual grading by Point Picker plug-in of Image J ( http://bigwww.epfl.ch/thevenaz/pointpicker/ ) and (2) automated grading by the Image J Particle Analysis algorithm ( http://imagej.net/Particle_Analysis ). The automated and manual AC cell counts were correlated with the Standardization of Uveitis Nomenclature score. RESULTS: The average numbers of AC inflammatory cells counted by the automated method were 8 ± 4.0, 18 ± 3.0, 42 ± 14.0, 81 ± 32.0, 117 ± 57.0, and 275 ± 67.0 cells/mm2 for grades 0, 0.5 + , 1 + , 2 + , 3 + , and 4 + , respectively. For the same clinical categories, the average manual cell counts were 6 ± 4.0, 18 ± 3.0, 34 ± 14.0, 72 ± 32.0, 92 ± 43.0, and 168 ± 65.0 cells/mm2, respectively. Zero cells were detected in the AC of healthy eyes. The automated and manual methods were highly correlated (R = 0.98, p < 0.001) and showed good correlation with the clinical grading (R = 0.88, p < 0.001). A mean AC particle size of 117.4 ± 108.8 µm was obtained by the automated method. CONCLUSIONS: Quantification of the AC cells imaged by SS AS-OCT shows good correlation with categorical clinical severity assessments in uveitis eyes. This approach may provide a more objective method for monitoring uveitis and response to uveitis therapy.

Evaluation of the corneal epithelium in non-Sjögren's and Sjögren's dry eyes: an in vivo confocal microscopy study using HRT III RCM.

BACKGROUND: The corneal epithelium is directly affected in dry eye syndrome. Thus, we attempted to describe the morphological features and evaluate the cellular density within the corneal epithelial layers in patients with non-Sjögren's (NSDE) and Sjögren's syndrome dry eyes (SSDE) by in vivo confocal microscopy (IVCM). METHODS: Central cornea was prospectively imaged by IVCM in 68 clinically diagnosed aqueous tear-deficient dry eyes and 10 healthy age-matched control eyes. Morphological characteristics of corneal epithelial layers and cellular densities were evaluated by four trained graders from the Doheny Eye Institute. RESULTS: Corneal epithelium in dry eyes presents morphological changes such as areas of enlarged and irregular shaped cells. In comparison with controls, the density of superficial epithelial cells was decreased in both the NSDE (P < 0.05) and SSDE groups (P < 0.01); the density of the outer layer of wing cells was smaller but not significantly different in NSDE (P > 0.05), but was lower in the SSDE group (P < 0.01); the density of the inner layer of wing cells was decreased in both the NSDE (P < 0.05) and SSDE groups (P < 0.01) and the density of basal epithelial cells was lower in both the NSDE (P < 0.01) and SSDE groups (P = 0.01). For all cell counts, the interclass correlation coefficient showed good agreement between graders (ICC =0.75 to 0.93). CONCLUSIONS: IVCM represents a reliable technique for examining the corneal epithelial microstructural changes associated with dry eyes, as well as for objectively and reproducibly quantifying cell densities within all corneal epithelial layers.

Comparison of manual & automated analysis methods for corneal endothelial cell density measurements by specular microscopy / Comparación de los métodos de análisis manual y automatizado para medir la densidad celular endotelial corneal mediante microscopio especular

Purpose: To determine the reliability of corneal endothelial cell density (ECD) obtained by automated specular microscopy versus that of validated manual methods and factors that predict such reliability. Methods:Sharp central images from 94 control and 106 glaucomatous eyes were captured with Konan specular microscope NSP-9900. All images were analyzed by trained graders using Konan CellChek Software, employing the fully- and semi-automated methods as well as Center Method. Images with low cell count (input cells number <100) and/or guttata were compared with the Center and Flex-Center Methods. ECDs were compared and absolute error was used to assess variation. The effect on ECD of age, cell count, cell size, and cell size variation was evaluated. Results:No significant difference was observed between the Center and Flex-Center Methods in corneas with guttata (p = 0.48) or low ECD (p = 0.11). No difference (p = 0.32) was observed in ECD of normal controls < 40 yrs old between the fully-automated method and manual Center Method. However, in older controls and glaucomatous eyes, ECD was overestimated by the fully-automated method (p = 0.034) and semi-automated method (p = 0.025) as compared to manual method. Conclusion: Our findings show that automated analysis significantly overestimates ECD in the eyes with high polymegathism and/or large cell size, compared to the manual method. Therefore, we discourage reliance upon the fully-automated method alone to perform specular microscopy analysis, particularly if an accurate ECD value is imperative

Objetivo: Determinar la fiabilidad de la densidad celular endotelial corneal (ECD) obtenida mediante microscopio especular automático frente a métodos manuales validados y factores predictivos de la fiabilidad. Métodos: Se capturaron imágenes nítidas de 94 controles y 106 ojos glaucomatosos con un microscopio especular Konan NSP-9900. Todas las imágenes fueron analizadas por examinadores expertos mediante el software Konan CellChek, utilizando los métodos automatizado total, semiautomático y de centrado. Se compararon las imágenes con bajo recuento celular (número de células <100) y/o córnea guttata con el método de centrado y centrado flexible. Se compararon las ECD, utilizándose el error absoluto para valorar la variación. Se evaluó el efecto de la ECD sobre la edad, el recuento celular, el tamaño celular y la variación del tamaño celular. Resultados: No se observó diferencia significativa entre los métodos de centrado y centrado flexible en las córneas con guttata (p = 0,48) o baja ECD (p = 0,11). No se observó diferencia (p = 0,32) en cuanto a ECD en los controles normales < 40 años entre el método totalmente automatizado y el método de centrado manual. Sin embargo, en los controles mayores y en los ojos glaucomatosos, la ECD fue sobreestimada por el método totalmente automatizado (p = 0,034) y el método semiautomático (p = 0,025), en comparación al método manual. Conclusión: Nuestros hallazgos muestran que los análisis automatizados sobreestiman considerablemente la ECD en los ojos con alto polimegatismo y/o gran tamaño celular, en comparación al método manual. Por tanto, no recomendamos confiar en el método totalmente automatizado por sí solo para realizar estudios mediante microscopio especular, particularmente en casos en que la precisión del valor de ECD sea imperativo

Comparison of manual & automated analysis methods for corneal endothelial cell density measurements by specular microscopy.

PURPOSE: To determine the reliability of corneal endothelial cell density (ECD) obtained by automated specular microscopy versus that of validated manual methods and factors that predict such reliability. METHODS: Sharp central images from 94 control and 106 glaucomatous eyes were captured with Konan specular microscope NSP-9900. All images were analyzed by trained graders using Konan CellChek Software, employing the fully- and semi-automated methods as well as Center Method. Images with low cell count (input cells number <100) and/or guttata were compared with the Center and Flex-Center Methods. ECDs were compared and absolute error was used to assess variation. The effect on ECD of age, cell count, cell size, and cell size variation was evaluated. RESULTS: No significant difference was observed between the Center and Flex-Center Methods in corneas with guttata (p=0.48) or low ECD (p=0.11). No difference (p=0.32) was observed in ECD of normal controls <40 yrs old between the fully-automated method and manual Center Method. However, in older controls and glaucomatous eyes, ECD was overestimated by the fully-automated method (p=0.034) and semi-automated method (p=0.025) as compared to manual method. CONCLUSION: Our findings show that automated analysis significantly overestimates ECD in the eyes with high polymegathism and/or large cell size, compared to the manual method. Therefore, we discourage reliance upon the fully-automated method alone to perform specular microscopy analysis, particularly if an accurate ECD value is imperative.

Comparison of Noncontact Specular and Confocal Microscopy for Evaluation of Corneal Endothelium.

PURPOSE: To compare endothelial cell analysis obtained by noncontact specular and confocal microscopy, using the Konan NSP-9900 and Nidek ConfoScan4 systems, respectively. METHODS: Three groups including 70 healthy eyes, 49 eyes with Fuchs endothelial corneal dystrophy (FECD), and 78 eyes with glaucoma were examined with both the Konan NSP-9900 specular microscope and the Nidek ConfocScan4 confocal microscope. Certified graders at the Doheny Image Reading Center compared corneal endothelial images from both instruments side by side to assess image quality. Endothelial cell density (ECD) measurements were calculated and compared using three different modalities: (1) each instrument's fully automated analysis; (2) each instrument's semiautomatic analysis with grader input; and (3) manual grading methods by certified grader. RESULTS: All normal eyes yielded gradable endothelial images, and most but not all glaucomatous eyes yielded images with high enough image quality to allow grading. In addition, in corneas with severe FECD, poor image quality precluded ECD grading by specular microscopy in 20 eyes (40.8%) but in only 4 (8.2%) confocal images from the same eyes. For the gradable images, the ECD values obtained using the manual grading method from either device were comparable with no statistically significant difference (P>0.05) between specular and confocal devices. Machine-generated ECD values were significantly different from manual results, measuring greater in all cases with specular microscopy. Machine-generated ECD values from confocal microscopy also differed significantly from manual determinations, but not in a consistent direction. Semiautomatic methods for both instruments obtained clinically acceptable ECD values. CONCLUSIONS: Automatic machine-generated ECD measurements differed significantly from manual assessments of corneal endothelium by both specular and confocal microscopy, suggesting that automated results should be used with caution. But ECD values derived manually were comparable between the two devices in both normal and glaucomatous eyes, suggesting that manually graded images from the two instruments can be used interchangeably for reliable ECD measurements. Because of a higher proportion of gradable images, confocal microscopy may be superior to specular microscopy for ECD measurements in FECD.

Validity of a new comprehensive pterygia grading scale for use in clinical research and clinical trial.

PURPOSE: To determine the reliability and agreement of a new comprehensive pterygium grading scale for use in clinical research and clinical trials. METHODS: Thirty eyes with pterygia were enrolled in this study. Primary gaze position and lateral gaze position images were taken of each eye with a modified single-lens reflex camera system. Our grading scale includes five parameters: two hyperemia parameters of pterygia on two different gaze position images and three size parameters, quantifying length, width, and area of the cornea encroachment of pterygium, using ImageJ software. All images were graded on the five parameters by two masked, certified reading center graders. Two graders independently graded all the images to determine inter-grader reliability. One grader regraded the images after 3 days to determine intra-grader reliability. Intraclass correlation coefficient (ICC) and inter-rater agreement statistic (κ) calculations were performed. RESULTS: The intra-grader reliability for hyperemia grading was high on both primary and lateral gazing positions (κ value is 0.93 and 0.96). The inter-grader reliability for hyperemia grading was also good (κ value is 0.85 and 0.87). The mean value of width, length, and area of the cornea encroachment of pterygium was 4.31 ± 2.04 mm, 2.08 ± 1.43 mm, and 7.84 ± 7.62 mm2, respectively. The intra-grader agreement on width, length, and area were excellent, with ICCs of 0.98 (95% CI 0.96-0.99), 0.99 (95% CI 0.98-1.0), and 0.97 (95% CI 0.94-0.99), respectively. The inter-grader agreement on width, length, and area were also excellent, with ICCs of 0.96 (95% CI 0.90-0.98), 0.99 (95% CI 0.98-0.99), and 0.99 (95% CI 0.97-0.99), respectively. CONCLUSIONS: There was excellent intra- and inter-observer reproducibility with the new comprehensive grading scale. This scale could lead to the development of standardized grading assessments and quantification of pterygia that would be valid in clinical research and clinical trials.

Corneal features in ocular graft-versus-host disease by in vivo confocal microscopy.

PURPOSE: The purpose of our study was to determine the morphological features of the corneal epithelial layers, sub-basal nerve plexus and anterior stroma in patients with ocular graft-versus-host disease (oGVHD) compared to non-GVHD dry eyes and normal controls, using in vivo confocal microscopy (IVCM). METHODS: IVCM was used to capture central cornea images from eight volunteers with normal healthy eyes, ten patients with non-GVHD dry eye syndrome (DES) and 15 patients with clinically diagnosed oGVHD, in a cross-sectional study. Morphological changes of the corneal epithelial layers and anterior stroma, characteristics of corneal nerves and presence of dendritic cells (DCs) were then evaluated. RESULTS: IVCM images obtained from 66 eyes were analyzed. The density of superficial epithelial cells was 636.07 ± 101.05 cells/mm2 in the oGVHD group, 827 ± 99.62 cells/mm2 in the DES group and 1277.2 ± 121.42 cells/mm2 in the control group (P < 0.001). The density of wing cells was 4499.79 ± 976.36 cells/mm2 in the oGVHD group, 4662.85 ± 319.72 cells/mm2 in DES group and 6556.38 ± 503.99 cells/mm2 in the control group (p < 0.001). The density of basal cells was 7850.93 ± 723.51 cells/mm2 in the oGVHD group, 8570 ± 913.32 cells/mm2 in DES group and 9759.8 ± 251.99 cells/mm2 in the control group (p < 0.01). The density of nerve fibers was 11.22 ± 5.46 mm/mm2 in the oGVHD group, 14.50 ± 4.27 mm/mm2 in DES group and 19.56 ± 4.75 mm/mm2 in the control group (p < 0.01). The DC density was 67.88 ± 71.82 cells/mm2 in the oGVHD group, 40.06 ± 31.95 cells/mm2 in the DES group and 29.45 ± 8.1 cells/mm2 in the control group (P > 0.05). Visible networks of activated keratocytes were seen in the anterior stroma of eyes with oGVHD and DES, but not in normal controls. CONCLUSIONS: IVCM revealed distinct microstructural changes in the corneas of patients with oGVHD and DES, similar between the two groups. Our findings suggest implications for use of IVCM to evaluate and monitor patients with dry eyes associated or not with GVHD.

Comparison of the Center and Flex-Center Methods of Corneal Endothelial Cell Analysis in the Presence of Guttae.

PURPOSE: To compare endothelial cell analysis by the center and flex-center methods in corneas with guttae of differing severity and to determine the minimum countable cell number for using only the flex-center method. METHODS: Forty-seven eyes with corneal guttae and 50 age-matched normal controls were enrolled in the study. Three images were captured in each central cornea with the noncontact specular microscope. Endothelial cell density (ECD), coefficient of variation (CV), and percentage of hexagonal cells (HEX) were analyzed by trained graders, using both center and flex-center methods. RESULTS: Consistent ECD and HEX values were obtained in normal eyes by both methods (P > 0.05). In corneas with guttae, ECD values obtained by the center method were 2.4% higher than those obtained with the flex-center method (P < 0.001). ECD values derived by both methods disagreed only when <30 cells were identified or <20 cells were analyzed. CV values obtained by the center method were 17.1% (P < 0.001) lower than those obtained by the flex-center method. HEX values obtained with both methods (P > 0.05) agreed. Regardless of guttae density, the ECD, CV, and HEX values of 3 images of each eye were in agreement (P > 0.05). CONCLUSIONS: In corneas with guttae, both center and flex-center methods can reliably determine ECD. Although current practice recommends the center method when at least 100 cells can be counted, our study suggests that the center method can provide a reliable ECD value when there are ≥30 contiguous countable cells in a central cornea endothelial image. The flex-center method is recommended when <30 contiguous cells are identified.

Correlation between corneal innervation and inflammation evaluated with confocal microscopy and symptomatology in patients with dry eye syndromes: a preliminary study.

PURPOSE: To evaluate corneal innervation and inflammatory cell infiltration using in vivo confocal microscopy (IVCM) and to correlate these findings with subjective symptoms of dry eye, as measured by the Ocular Surface Disease Index (OSDI) in patients with non-Sjögren's (NSDE) and Sjögren's syndrome dry eyes (SSDE). METHODS: Central corneal images were prospectively captured from 10 age-matched healthy control eyes, 24 eyes with clinically diagnosed NSDE and 44 eyes with clinically diagnosed SSDE, using IVCM (HRT III RCM). Density, tortuosity and reflectivity of corneal nerves, presence of inflammatory dendritic cells (DCs) and OSDI scores were evaluated. RESULTS: Images obtained by IVCM from 78 eyes were analyzed. The density of nerve fibers was 1562 ± 996 µm/frame in the SSDE group, 2150 ± 1015 µm/frame in the NSDE group and 2725 ± 687 µm/frame in the control group (P < 0.05, ANOVA). In comparison to the control group, the density of nerve fibers was decreased in the SSDE (P < 0.001) and the NSDE groups (P = 0.06), with increased nerve tortuosity and decreased reflectivity in both groups (both P < 0.05). The density of DCs was 71.65 ± 72.54 cells/mm2 in the SSDE group, 40.33 ± 31.63 cells/mm2 in the NSDE group and 27.53 ± 5.58 cells/mm2 in the control group (P < 0.05, ANOVA). In comparison to the control group, the density of DCs was increased in the SSDE (P < 0.001) and the NSDE groups (P = 0.07). Significant correlations were found between the nerve density and DC density (r = -0.57, P < 0.001), between the nerve density and OSDI scores (r = -0.91, P < 0.001) and between the nerve reflectivity and OSDI scores (r = -0.75, P < 0.001). CONCLUSIONS: The corneas of eyes affected with NSDE and SSDE are characterized by alterations in corneal innervation and infiltration of inflammatory DCs. Corneal nerve density and reflectivity are correlated with severity of subjective dry eye symptoms, as measured by OSDI score.

Quantitative Analysis of Depth, Distribution, and Density of Cysts in Acanthamoeba Keratitis Using Confocal Microscopy.

PURPOSE: To quantify the density, distribution, and depth of invasion of cysts in the corneas of eyes with acanthamoeba keratitis (AK) by in vivo confocal microscopy (IVCM) with a novel scanning pattern. METHODS: The medical records of patients with AK evaluated at the Doheny Eye Center UCLA between September 2014 and July 2016 were reviewed retrospectively. Patients with clinically diagnosed AK underwent IVCM at various time points during their clinical course. Five corneal locations were scanned at each time point: the central area and 4 standard points on the peripheral cornea corresponding to temporal, nasal, inferior, and superior locations. The IVCM scans were manually graded to quantify the maximum depth of invasion and density of cysts. RESULTS: Twenty-one eyes of 18 patients with visible cysts on IVCM were included. Mean cyst density at presentation was 214.1 ± 120.2/mm (range: 64-484 cells/mm), and the average cyst depth was 164.3 ± 81.2 µm (range: 17-290 µm). In 17 eyes, the average cyst depth was 139.4 ± 68.6 µm (range: 17-245 µm), mean cyst density was 177.9 ± 99.6/mm, and an average of 1.4 ± 1.3 quadrants was infiltrated at presentation, and reached clinical resolution with medical treatment without surgical intervention. Four eyes that ultimately underwent therapeutic penetrating keratoplasty had cysts in all 4 quadrants and deeper cyst infiltration; the average cyst depth in these corneas was 270.5 ± 17.5 µm (range: 252-290). CONCLUSIONS: Eyes with AK requiring therapeutic keratoplasty were more likely to have a deeper and more diffuse penetration of cysts in the cornea compared with those resolving with medical treatment.

Characterization of Corneal Involvement in Eyes With Mucous Membrane Pemphigoid by In Vivo Confocal Microscopy.

PURPOSE: To describe the morphological features of the corneal epithelial layers, subbasal nerve plexus, stroma, and endothelium in patients with mucous membrane pemphigoid (MMP) as shown by in vivo confocal microscopy (IVCM). METHODS: Central corneal images were captured from 10 healthy age-matched control eyes and 30 eyes with clinically diagnosed MMP by in vivo laser scanning confocal microscopy (HRT III RCM). Morphological changes of the corneal epithelial layers, stroma, and endothelium, characteristics of corneal nerves, and presence of inflammatory dendritic cells (DCs) were evaluated. RESULTS: Images obtained by IVCM from 40 eyes were analyzed. The eyes with MMP were divided into 2 groups based on clinical staging: 16 eyes with end-stage MMP and 14 eyes with non-end-stage MMP. Compared with controls, IVCM in eyes with end-stage MMP displayed severe conjunctivalization and neovascularization of the cornea, with otherwise limited identifiable cellular or structural elements. Those with non-end-stage MMP showed metaplasia of the corneal epithelial layers, presence of hyperreflective cells similar to conjunctival cells, intraepithelial defects, fibrosis of anterior stroma, and hyperreflective endothelial deposits. Images of the subbasal nerve plexus demonstrate significant reduction in density (1251.3 ± 806.9 µm/frame vs. 2688.8 ± 607.33 µm/frame, P < 0.001), increased tortuosity (2.76 ± 0.6 vs. 2.3 ± 0.42, not significant), decreased reflectivity (2.73 ± 0.4 vs. 3.46 ± 0.52, P < 0.01), and increased density of DCs (115 ± 88 cells/mm vs. 43.9 ± 28.14 cells/mm, P < 0.05) in MMP-affected eyes compared with controls. CONCLUSIONS: IVCM reveals profound and variable microstructural changes in the corneas of patients with MMP compared with normal controls. Our study demonstrated decreased corneal nerve density and elevated DC density in eyes with non-end-stage MMP compared with normal controls. Frequent scarring, conjunctivalization, and neovascularization observed in eyes with end-stage MMP preclude recognition by IVCM of the morphologic architecture of the corneal layers. Our findings suggest implications for using IVCM to evaluate and monitor patients with MMP.