Retinal autofluorescence findings after COVID-19.

The main purpose of this study was to investigate the presence of retinal autofluorescence findings in COVID-19 patients. Observational study conducted in São Paulo in 2020. Demographic, medical history, and concomitant events, as well as medications used, hospitalization details, and laboratory test results, were obtained. Patients underwent eye examination and multimodal imaging, including color, red-free, autofluorescence fundus photography and optical coherence tomography. Eighteen patients had autofluorescence findings (6 females; average age 54 years, range 31 to 86 years; 26 eyes). Hyper-autofluorescence findings were present in 6 patients, Hypo-autofluorescence in 14 patients, and 6 patients had mixed pattern lesions. Retinal autofluorescence abnormalities were present in COVID-19 patients and may be secondary to primary or secondary changes caused by the SARS-CoV-2.

Presumed SARS-CoV-2 Viral Particles in the Human Retina of Patients With COVID-19.

Importance: The presence of the SARS-CoV-2 virus in the retina of deceased patients with COVID-19 has been suggested through real-time reverse polymerase chain reaction and immunological methods to detect its main proteins. The eye has shown abnormalities associated with COVID-19 infection, and retinal changes were presumed to be associated with secondary microvascular and immunological changes. Objective: To demonstrate the presence of presumed SARS-CoV-2 viral particles and its relevant proteins in the eyes of patients with COVID-19. Design, Setting, and Participants: The retina from enucleated eyes of patients with confirmed COVID-19 infection were submitted to immunofluorescence and transmission electron microscopy processing at a hospital in São Paulo, Brazil, from June 23 to July 2, 2020. After obtaining written consent from the patients' families, enucleation was performed in patients deceased with confirmed SARS-CoV-2 infection. All patients were in the intensive care unit, received mechanical ventilation, and had severe pulmonary involvement by COVID-19. Main Outcomes and Measures: Presence of presumed SARS-CoV-2 viral particles by immunofluorescence and transmission electron microscopy processing. Results: Three patients who died of COVID-19 were analyzed. Two patients were men, and 1 was a woman. The age at death ranged from 69 to 78 years. Presumed S and N COVID-19 proteins were seen by immunofluorescence microscopy within endothelial cells close to the capillary flame and cells of the inner and the outer nuclear layers. At the perinuclear region of these cells, it was possible to observe by transmission electron microscopy double-membrane vacuoles that are consistent with the virus, presumably containing COVID-19 viral particles. Conclusions and Relevance: The present observations show presumed SARS-CoV-2 viral particles in various layers of the human retina, suggesting that they may be involved in some of the infection's ocular clinical manifestations.

COVID-19 Retinal Findings in Patients Admitted to Intensive Care Units and Wards.

The main purpose of this study was to investigate ocular clinical findings in patients with confirmed COVID-19 infection, of various levels of disease severity, who required mechanical ventilation and admission to intensive care units or specialized wards. Longitudinal, observational study conducted from March 2020 to June 2020. Color fundus and red-free photography were performed in both eyes following pupillary dilation. 104 participants were recruited from 2 different centers: 60 (58%) from the Hospital Municipal de Barueri intensive care unit (ICU) and 44 (42%) from specialized wards for patients with COVID-19 at the Hospital São Paulo. 21.9% presented with eye lesions, in 3% these lesions were vision compromising. Our results have shown similar rate of intraocular lesions in patients in both the ward or intensive care unit, regardless of medication use, including anticoagulant drugs.

Projection-Resolved Optical Coherence Tomography Angiography Parameters to Determine Severity in Diabetic Retinopathy.

Purpose: The purpose of this study was to assess projection-resolved optical coherence tomography angiography (PR-OCTA) vessel density (VD) and foveal avascular zone (FAZ) in determining severity within diabetic retinopathy (DR) and their accuracy in identifying high-risk DR patients. Methods: This was a retrospective study with 72 eyes of 52 DR patients, assessing the VD and FAZ area of the superficial capillary plexus (SCP) and deep vascular plexus (DVP), for both 3 × 3-mm and 6 × 6-mm scans between the DR groups (mild to moderate, severe and proliferative DR [PDR]). For accuracy, the severe and PDR groups were merged, representing the high-risk DR group for receiver operator characteristic analysis. VD of OCTA images with and without PR were compared. Results: In mild to moderate, severe, and PDR groups, there were 31, 21, and 20 eyes, respectively. PR-OCTA improved VD analysis only in the DVP and particularly in advanced DR stages (P = 0.042). In the 3 × 3-mm PR scans, all superficial and deep parameters were significantly different between severe and PDR groups (P ≤ 0.020), but only the mean VD of SCP and DVP was also significant between the mild to moderate and severe groups (P ≤ 0.007). In the 6 × 6-mm scans, the superficial VD, deep VD, and superficial FAZ were significantly different between the severe and PDR groups (P ≤ 0.029). The superficial VD and deep VD of the 3 × 3-mm scans were good parameters for detecting high-risk patients (area under the curve = 0.829 and 0.895, respectively). Conclusions: PR-OCTA improved VD analysis of DVP. The 3 × 3-mm SCP and DVP VD were the most accurate in detecting high-risk DR.

Survival analysis of conjunctival limbal grafts and amniotic membrane transplantation in eyes with total limbal stem cell deficiency.

PURPOSE: To evaluate the survival of conjunctival limbal grafts and amniotic membrane transplantation (AMT) for total limbal stem cell deficiency (LSCD) and the influence of several parameters as cause of LSCD, dry eye, keratinization, eyelid abnormalities, HLA compatibility, systemic immunosuppression, and keratoplasty (PKP) on surgical outcome. DESIGN: Prospective, noncomparative, interventional case series. METHODS: Thirty-three eyes of 31 patients with total LSCD that underwent conjunctival limbal grafts and AMT at the Department of Ophthalmology, Federal University of São Paulo were included in this study. Cumulative graft survival as well as the influence of several variables on surgical outcome was analyzed. RESULTS: Ten eyes (30%) underwent conjunctival limbal autograft and 23 (70%) underwent conjunctival limbal allograft from living HLA-matched donor. Graft survival was seen in 13 eyes (40%) at 1 year and in 11 eyes (33.3%) at 2 years, with a cumulative survival of 33% after a mean follow-up time of 33 months. Increase in postoperative visual acuity was observed in 20 eyes (60.6%) during this period. Marked impact on graft survival was observed for patients with Stevens-Johnson syndrome, dry eye, keratinization, eyelid abnormalities, and allogeneic conjunctival limbal transplantation (independently of HLA compatibility) (P < .05). Preoperative dry eye was the most important prognostic parameter for surgical outcome (P < .001). CONCLUSIONS: Conjunctival limbal grafts associated with AMT are useful for restoring corneal epithelium phenotype in eyes with total LSCD. However, the cumulative survival declines substantially over a 2-year period. Considering all investigated variables, dry eye was the most important prognostic parameter.

Clinical treatment of corneal infection due to Fonsecaea pedrosoi--case report.

UNLABELLED: To report an unusual case of fungus keratitis due to Fonsecaea pedrosoi that developed after corneal trauma. CASE REPORT: A 18-year-old male presented with a corneal ulcer in the right eye, 28 days after a trauma with glass fragments. Corneal scrapings were collected for smears and culture. Dematiaceous hyphae were seen on wet mounts of the scrapings and dark pigmented colonies grew repetitively on the culture media; all colonies were identical, and were subsequently identified as Fonsecaea pedrosoi. Treatment was initiated with topical natamycin at one hour intervals, 200 mg oral ketoconazole per day and later changed to a combination of 200 mg ketoconazole and amphotericin B. In humid tropical regions Fonsecaea pedrosoi is one of the primary causes of human chronic cutaneous mycosis, chromoblastomycosis. Combination of systemic and topical antifungal medications may provide the best option for cure in corneal chromoblastomycosis.

Clinical treatment of corneal infection due to Fonsecaea pedrosoi: case report

Relato de um caso atípico de infecção fúngica da córnea causada pelo microrganismo Fonsecaea pedrosoi após trauma ocular. Paciente, masculino, estudante de 18 anos, apresentou-se ao Setor de Doenças Externas Oculares do Departamento de Oftalmologia da UNIFESP com úlcera de córnea paracentral de 3,5 x 3,5 mm e aspecto branco-acinzentado com bordas infiltradas, 28 dias após trauma em ocular por vidro. Foi realizado raspado da córnea e o material enviado para análise microbiológica. Foi observado crescimento de colônias em meio de cultura e posteriormente colocadas em solução de lactofenol-azul de algodão. Verificou-se a presença de hifas dermáceas de pigmento escuro, identificado como Fonsecaea pedrosoi. Tratamento foi iniciado com natamicina 5% tópica a cada hora e cetoconazol 200 mg por dia. Subseqüentemente foi substituído pela combinação cetoconazol e anfotericina B. Fonsecaea pedrosoi é uma das principais causas em humanos de micose crônica cutânea, cromoblastomicose, em regiões úmidas tropicais. A combinação de antimicóticos sistêmicos e tópicos pode ser a melhor opção para pacientes no tratamento de cromoblastomicose da córnea.

Flap and stromal bed thickness in laser in situ keratomileusis enhancement.

PURPOSE: To evaluate whether flap thickness changes after the primary laser in situ keratomileusis (LASIK) procedure and to assess the accuracy of intraoperative pachymetry and ablation depth measurements in predicting stromal bed thickness before enhancement in eyes that have had primary myopic LASIK. SETTING: Bascom Palmer Eye Institute, Miami, Florida, USA. METHODS: This retrospective noncomparative interventional case series comprised 57 eyes of 42 patients who had LASIK enhancement between June 2001 and September 2002. Exclusion criteria included previous ocular surgery or complications during the first LASIK procedure. Only patients who had had LASIK and enhancement by the same surgeon at our institution and had intraoperative pachymetry readings for both procedures were included. The original flap was relifted in all enhancement procedures. Corneal thickness was routinely measured intraoperatively by ultrasound pachymetry. The age, eye, refraction, date of primary LASIK, central corneal thickness (CCT) and central stromal bed thickness at primary LASIK, depth of ablation, flap thickness (subtraction pachymetry), date of enhancement, CCT and central stromal bed thickness at enhancement, and flap thickness at enhancement were recorded. RESULTS: Thirty-one eyes of 26 patients were myopic and 26 eyes of 16 patients were hyperopic before primary LASIK. The mean time between LASIK and enhancement was 218 days +/- 115 (SD) (193 +/- 88 days in myopic eyes and 248 +/- 136 days in hyperopic eyes [P = .068]). The flap tended to be thicker at enhancement than in the primary LASIK procedure by 9.3 +/- 25.7 microm in myopic eyes (P = .054) and 10.5 +/- 16.6 microm in hyperopic eyes (P = .004). A strong correlation was found between flap thickness in the first and second procedures in myopic and hyperopic eyes (r = 0.6). In myopic eyes, the mean difference between the estimated stromal bed thickness after the first procedure (central bed thickness- ablation depth) and the stromal bed thickness measured directly at enhancement was not statistically significant (3 +/- 29 microm; P = .54, paired t test). A strong correlation was found between the 2 measurements (r = 0.8, P<.001). Another strong correlation was found in myopic eyes between the estimated corneal thickness after the primary LASIK and the corneal thickness measured at enhancement (r = 0.81, P<.001). No correlation was found between the difference in flap thickness and the time to enhancement (r = 0.09 in myopic eyes and r = 0.01 in hyperopic eyes). CONCLUSIONS: Flap thickness tended to be thicker at enhancement than at primary LASIK. Intraoperative pachymetry and ablation depth measurements proved to be precise tools to predict stromal bed thickness before enhancement in eyes that had had primary myopic LASIK. This information may help in planning LASIK enhancements.

Corneal flap thickness in laser in situ keratomileusis using the Moria M2 microkeratome.

PURPOSE: To determine the predictability of flap thickness in laser in situ keratomileusis (LASIK) using the Moria M2 microkeratome and identify factors that may be related to variations in flap thickness. SETTING: Laser Vision Correction Center, Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, Florida, USA. METHODS: Charts of 208 patients having same-day bilateral LASIK using the Moria M2 microkeratome were reviewed. Intraoperative pachymetry was performed routinely. The right eye was always treated first. The same suction ring, stop, microkeratome head (110 microm or 130 microm), and blade were used in fellow eyes. Subtraction pachymetry was used to calculate flap thickness. Other collected data included age, keratometry, corneal diameter, and preoperative spherical equivalent (SE). RESULTS: With the 110 microm head and slow translation velocity in both eyes, the mean flap thickness was 151.6 microm +/- 24.0 (SD) and 148.5 +/- 24.3 microm in the right and left eyes, respectively. With the 110 microm head and fast translation velocity in both eyes, the mean thickness was 136.2 +/- 25.5 microm and 132.8 +/- 23.5 microm, respectively. With the 130 microm head and fast translation velocity, the mean flap thickness was 145.8 +/- 25.4 microm and 139.9 +/- 25.5 microm, respectively. Flaps were thinner with fast translation velocity, the 110 microm head, and presumably duller blades used in the left eyes. There was a weak but statistically significant inverse correlation between flap thickness and age and between flap thickness and SE. A stronger correlation was found in flap thickness between right and left eyes. CONCLUSIONS: Flap thickness with the Moria M2 microkeratome was variable. Fast translation velocity, a used (presumably duller) blade, and the 110 microm head produced thinner flaps. Given the potential variation in flap thickness (SD 23.5 to 25.5 microm), intraoperative pachymetry might be an adjunctive measure to prevent residual stromal beds that are thinner than planned, especially in patients with high myopia and/or thin corneas.

Corneal infections after implantation of intracorneal ring segments.

PURPOSE: To report risk factors, clinical course, and outcome in patients with infectious keratitis following implantation of intracorneal ring segments (ICRS). METHODS: The records of 8 patients with culture-proven infectious keratitis after ICRS (Ferrara or Intacs) implantation were retrospectively reviewed. Age, gender, corneal findings, ocular abnormalities, the condition that led to ICRS implantation, immediate prior use of a contact lens, elapsed time between implantation and the onset of symptoms, previous medications, and systemic disorders were noted. RESULTS: Culture-positive infectious keratitis developed in 7 eyes of 7 patients (2 men and 5 women) with a mean age of 35 years who underwent Ferrara implantation for the treatment of keratoconus and in a 29-year-old man who underwent Intacs implantation for correction of low myopia. Contact lens use, diabetes, and trauma were factors possibly associated with the risk of infection in three cases. Microorganisms, identified in all cases, included Staphylococcus aureus, Streptococcus viridans, Streptococcus pneumoniae, Pseudomonas sp, Nocardia sp, Klebsiella sp, and Paecylomices sp. Onset of symptoms of infection varied from less than 1 week to 22 months postoperatively, depending on the infecting organism. CONCLUSIONS: Infectious keratitis following ICRS implantation is a sight-threatening complication for which early recognition and rapid institution of appropriate treatment may result in a better visual outcome.

Ciprofloxacin and levofloxacin resistance among methicillin-sensitive Staphylococcus aureus isolates from keratitis and conjunctivitis.

PURPOSE: The majority of Staphylococcus aureus isolated from ocular infections are methicillin sensitive (MSSA). Fluoroquinolone resistance among methicillin- resistant Staphylococcus aureus is well known but not among MSSA. In this study we investigate the emergence of fluoroquinolone (ciprofloxacin, levofloxacin) resistance among methicillin-sensitive Staphylococcus aureus isolates from keratitis and conjunctivitis. DESIGN: Cross-sectional study. METHODS: Minimal inhibitory concentration susceptibility profiles for 1230 S. aureus isolates from keratitis and conjunctivitis were analyzed. Historical and current rates of emerging fluoroquinolone resistance for methicillin-resistant (MRSA) and methicillin-sensitive isolates (MSSA) were evaluated. Sensitivity patterns for vancomycin and gentamicin were also documented. RESULTS: Ciprofloxacin resistance among corneal and conjunctival S. aureus isolates during the first 6 years after its introduction (baseline) (1990-1995) was 8% and ranged from 3% to 11%. The rate for the most recent 6 years (current) (1996-2001) was 20.7%. Baseline ciprofloxacin resistance for MSSA isolates was 2% vs 55.8% for MRSA. The current resistance rate for MSSA is 5% vs 83.7% for MRSA. The general resistance rate from January 2000 to December 2001 for levofloxacin was 25.5% vs 33.2% for ciprofloxacin (P =.13). The levofloxacin-resistance rate for MSSA was 4.7% vs 11.9% for ciprofloxacin (P =.05). In MRSA isolates, a higher resistance rate was found for ciprofloxacin (95.7%) vs levofloxacin (82.1%) (P =.04). No resistance for vancomycin was documented for any of the S. aureus isolates. Gentamicin susceptibility for MSSA was 99% and MRSA was 86%. CONCLUSIONS: Ciprofloxacin and levofloxacin resistance among methicillin-sensitive S. aureus corneal and conjunctival isolates is increasing. This is of concern because it narrows treatment choices for the management of these common ocular infections.

Bilateral Candida parapsilosis interface keratitis after laser in situ keratomileusis.

We report a case of Candida infection after laser in situ keratomileusis (LASIK) and review the literature for reports of post-LASIK fungal infections. Risk factors may include postoperative surgical intervention and extended use of topical steroids.

Different cell sizes in human limbal and central corneal basal epithelia measured by confocal microscopy and flow cytometry.

PURPOSE: In the epidermis, the highest clonogenicity, a feature of stem cells (SCs), is found in the smallest keratinocyte. In the limbal-corneal (LC) epithelium the SCs are exclusively localized in the basal epithelial layer of the limbal domain. The current study was conducted to determine whether this spatial SC arrangement is reflected in differences in the cell size between limbal and corneal cells. METHODS: In vivo confocal microscopy was used to scan and measure the size of the cells of the central cornea and the superior limbus in five normal subjects, from the superficial to the basal cell layer. Limbal and corneal pure epithelial sheets were isolated by dispase digestion from human tissues and dissociated into single cells by trypsin digestion. The forward (FSC; a relative measure of cell size) and side (SSC; a relative measure of cytoplasmic complexity) light-scattering properties of these cells were determined by flow cytometry. RESULTS: Confocal microscopy showed that diameters of the basal cells of the limbal and corneal zones were 10.1 +/- 0.8 and 17.1 +/- 0.8 micro m, respectively. The corresponding values for the superficial layers were 19.9 +/- 1.6 and 36.6 +/- 1.6 microm, respectively (P < 0.0001). The mean FSC and SSC of the limbal cells amounted to 65.7% +/- 8.7% and of the corneal cells, 74.4% +/- 4.6%. Furthermore, only 1.40% +/- 0.83% and 0.69% +/- 0.37% of the corneal cells had FSC and SSC equal to the lowest 15% of FSC and SCC of the limbal cells, respectively, indicating that the limbus contained a substantial proportion of very low FSC and SSC cells for which there was no corneal counterpart. CONCLUSIONS: The data collectively demonstrate that the smallest cells are located in the limbal basal epithelium. This feature may help isolate corneal SCs located in the limbus.

Novel enzymatic isolation of an entire viable human limbal epithelial sheet.

OBJECTIVE: To develop a reproducible method of isolating an intact viable human limbal epithelial sheet. METHODS: Human pigmented limbus was incubated at 4 degrees C for 18 hours in supplemental hormonal epithelial medium (SHEM) containing 50 mg/mL dispase II and 100 mM sorbitol. A loose limbal epithelial sheet was separated by a spatula. The remaining stroma was digested and subcultured. The viability of isolated cells was assessed. Isolated epithelial sheets and remaining stroma were subjected to immunostaining. Sheets 1.5 mm in length were cultured in SHEM on plastic until confluence, and cell extracts were subjected to Western blot analysis. RESULTS: Intact limbal epithelial sheets were consistently isolated. Pigmented palisades of Vogt revealed large superficial squamous cells and small basal cuboidal cells. No epithelial cells grew from the remaining stroma. Mean viability was 80.7% +/- 9.1%. The basal epithelium was negative to keratin 3 and connexin 43, but was scatter positive for p63. The epithelial sheet showed negative staining for laminin 5 and collagen VII, but interrupted linear basal staining for collagen IV. The remaining stroma showed negative staining for laminin 5, positive linear staining for collagen IV in the basement membrane, and diffuse staining for collagen VII in the superior stroma subjacent to the basement membrane. Western blot analysis revealed that cells originating from the limbal sheets expressed keratin 3 and p63. CONCLUSIONS: An intact limbal epithelial sheet can be consistently and reproducibly isolated and contains stem cell characteristics in the basal epithelium by degrading laminin 5 and part of collagen IV, and disassembling collagen VII.

Cleavage of corneal basement membrane components by ethanol exposure in laser-assisted subepithelial keratectomy.

PURPOSE: To determine the anatomic cleavage plane after exposure to 20% ethanol for approximately 20 to 25 seconds to create an epithelial flap in laser-assisted subepithelial keratectomy (LASEK). SETTING: Ocular Surface Research & Education Foundation, Miami, Florida, and Hermann Eye Center Refractive Surgery Center, Houston, Texas, USA. METHODS: Immunofluorescence staining using monoclonal antibodies against laminin 5, collagen VII, and integrins beta(1) and beta(4) was performed to determine the anatomic location of the cleavage plane in an epithelial flap created by 20-second exposure to 20% ethanol in cadaver eyes and in epithelial flaps obtained from LASEK patients. RESULTS: Immunofluorescence staining to laminin 5 and integrin beta(4) was patchy in the lifted flap and the remaining corneal basement membrane. Immunostaining to collagen VII, the main component of anchoring fibrils, remained exclusively in the corneal bed. Immunostaining to integrin beta(1), present in the pericellular location of all epithelial cell layers, remained exclusively in the epithelial flap. This finding was consistent in cadaver corneas and LASEK epithelial flaps. CONCLUSIONS: The cleavage plane of the ethanol-induced corneal epithelial flap is located between the lamina lucida and the lamina densa of the basement membrane, where integrin beta(4) interacts with laminin 5 to form hemidesmosomes.

Idiopathic limbal stem cell deficiency.

PURPOSE: To describe and to characterize the clinical findings and prognosis of patients with idiopathic limbal stem cell deficiency (LSCD). DESIGN: Retrospective case series. PARTICIPANTS/METHODS: We reviewed records from seven patients whose LSCD had been diagnosed clinically and confirmed by impression cytology and in whom the cause of LSCD was never identified. A detailed history, clinical records, and results of slit-lamp biomicroscopy, photography, vital staining, and impression cytology were evaluated. RESULTS: Six of seven patients (86%) were women, indicating a female predominance. Two patients were from the same family, whereas one other had a positive family history. Severe photophobia was noted in all patients and reduced vision in three patients. The main clinical findings included superficial vascularization, worse in the superior followed by the inferior and nasal cornea. The limbal regions showed a loss of limbal palisades of Vogt, and the adjacent peripheral cornea revealed an irregular and hazy epithelium with positive late fluorescein staining and the presence of conjunctival goblet cells by impression cytology. LSCD was bilateral in all patients but asymmetric in four. During a mean follow-up of 6.1+/- 3.8 years, the visual acuity decreased in both eyes of one patient after cataract extraction and in both eyes of two other patients without surgery. The process of conjunctivalization advanced in four patients (57%) and remained stable in three (43%) without surgical intervention. CONCLUSIONS: Idiopathic LSCD is a rare and as yet poorly recognized clinical entity, and the findings reported herein may help explore how progressive loss of limbal stem cells occurs. Correct diagnosis of idiopathic LSCD is important so that the patient will not be subjected to unnecessary surgeries, which may actually severely worsen the clinical course.