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.

Diffuse uveitis and chorioretinal changes after yellow fever vaccination: a re-emerging epidemic.

BACKGROUND: With increasing incidence of yellow fever, mass campaign vaccinations are underway and little ophthalmological alterations have been reported in literature, specially regarding non-combined vaccines. CASE PRESENTATION: We report the case of a patient with no previous ocular or systemic diseases whom received a single dose of yellow fever vaccination and developed haematological, hepatic and renal alterations progressing with a later onset bilateral asymmetric diffuse uveitis. Ophthalmological findings included fine keratic precipitates scattered throughout the cornea and mild vitritis. Multimodal evaluation showed subtle puntiform choriocapillaris changes with decreased vascular density associated. The patient had a good visual outcome after mild oral prednisone dose, but the image findings have not presented remission. CONCLUSIONS: Clinicians should be aware of clinical and subclinical ocular manifestations such as subtle puntiform choriocapillaris changes as possible vaccine-related adverse events with potential to impact vision.

Novel application of anterior segment optical coherence tomography for periocular imaging.

OBJECTIVE: This study aims to assess the use of optical coherence tomography (OCT) imaging for periocular skin lesions and to determine which characteristic features of these images can be correlated to histopathology. DESIGN: This is an ongoing prospective study with Research Ethics Board approval. PARTICIPANTS: Fifty patients over 18 years old with lesions clinically suspicious of nonmelanoma skin cancer on the periocular region were included in this study. METHODS: After consent was obtained, clinical photographs and dermatoscopic images were obtained (DermLite II Hybrid M) from the lesion and its contralateral side. Subsequently, the patient was subjected to OCT imaging using the anterior segment module of a spectral domain OCT (Optovue Avanti) and images of the contralateral skin were also obtained. Surgical excision of the lesion was performed and sent for histopathological examination as per routine treatment. OCT images were then correlated to their matching digitalized histopathology section (Philips Ultra Fast Scanner 1.6 RA). RESULTS: Based on the OCT images acquired from 50 patients, 8 predominant architectural features have been correlated to histopathology: hyperkeratosis, acanthosis, loss of dermal-epidermal junction delineation, hyporeflective tumour nests, cystic structures, "bunch of grapes" nodules, hyperreflective nests, and ulcerations. Results observed from 45 malignant lesions (basal cell carcinoma, squamous cell carcinoma, and sebaceous gland carcinoma) suggest that groups of features and their layout within the same OCT image may be associated to specific tumour characteristics. CONCLUSIONS: Current data suggest that anterior-segment OCT imaging is a noninvasive imaging modality for periocular lesions and may be a valuable tool to help differentiate between some tumour types before a biopsy is performed.

Optimizing optical coherence tomography and histopathology correlation in retinal imaging.

OBJECTIVE: To develop a methodology to correlate optical coherence tomography (OCT) images and histopathological sections from the same eye. Part 1: To determine the best fixative for optimal OCT and histopathological analysis in post-mortem eyes. Part 2: A protocol is proposed to correlate histopathological features and OCT scans from the same post-mortem eyes. DESIGN: Experimental study. PARTICIPANTS: Part 1: Twenty-three rabbit eyes and 14 post-mortem human eyes. Part 2: Nineteen post-mortem human eyes. METHODS: Part 1: Six different fixatives were tested, and specimens were evaluated on 4 criteria: globe shape, structure opacification, retinal detachment, and nuclear details. Part 2: Based on the findings from Part 1, fixed human eyes were imaged using OCT. Orientation-controlled histopathological processing was performed to obtain serial tissue sections from paraffin embedded tissue, which were matched to corresponding OCT images. RESULTS: Part 1: Of the 6 fixatives, 2% glutaraldehyde and Davidson's solution met the proposed criteria in rabbit eyes. Of these, glutaraldehyde showed similar results in human eyes and was selected for Part 2. Part 2: Using anatomical landmarks, cross-sectional histopathological sections in the same orientation as the OCT images were correlated to their corresponding OCT images. Retinal lesions such as a macular hole, an epiretinal membrane, and the presence of drusen were easily correlated, proving the reliability of our methodology. Moreover, the photoreceptor's inner/outer junction was correlated to a hyperreflective band on OCT. CONCLUSIONS: A standardized protocol was developed to correlate OCT images and histopathological findings by generating serial cross-sections of the retina, which can be used to better understand otherwise ambiguous OCT findings.

Novel treatment of papillomatous conjunctival lesions using pattern scanning laser photocoagulation: 1-Year results.

PURPOSE: To evaluate the safety and 1-year effect of pattern scanning laser photocoagulation treatment for pedunculated papillomatous and sessile conjunctival lesions in a low-resource setting with extremely limited operating room access. METHODS: Adult patients with clinical diagnosis of conjunctival papilloma underwent complete ophthalmologic exam including anterior segment photography. After topical anesthesia and toluidine blue 1% instillation, the lesion was treated by pattern scanning photocoagulation using a duration time that varied from 20 to 100 ms and power from 600 to 1800 mW, treating the entire lesion surface with a 2 mm margin. Patients were examined weekly for a month then monthly and underwent retreatment as necessary. RESULTS: Six patients and seven eyes that had clinically significant non-malignant pedunculated or sessile papillomatous lesions were treated. All lesions responded to treatment, with complete resolution after an average of 2.3 sessions. Procedures were well tolerated with only minor mild discomfort persisting up to two days post-treatment. Patients were followed for a mean follow-up time of 13 months with no recurrences reported. CONCLUSION: Short-term results of the pattern scanning laser photocoagulation approach, with toluidine blue for papillomatous conjunctival lesions are favorable with a 100% success rate in this cohort. This rate is comparable to surgical excision. This novel strategy proved to be a less resource intensive alternative that not only could demonstrate its usefulness in settings with chronic operating room shortages, but also in recurrent cases. Longer follow-ups with a larger sample size and cost-analysis are necessary to confirm our findings.

Optical Coherence Tomography Angiography of Chorioretinal Diseases.

Fluorescein angiography (FA) and indocyanine green angiography (ICGA) have been the gold standard for the evaluation of retinal and choroidal vasculature in the last three decades and have revolutionized the diagnosis of retinal and choroidal vascular diseases. The advantage of these imaging modalities lies in their ability to document retinal and choroidal vasculature through the dynamic assessment of contrast transit over time in the intravascular and extravascular spaces. However, disadvantages include the absence of depth resolution, blurring of details by contrast leakage, and the inability to selectively evaluate different levels of the retinal and choroidal microvasculature. In addition, these angiographic methods require intravenous dye, which may cause adverse reactions such as nausea, vomiting, and rarely, anaphylaxis. Optical coherence tomography angiography (OCTA) is a noninvasive imaging technique that, in contrast to dye-based angiography, is faster and depth-resolved, allowing in some cases for more precise evaluation of the vascular plexuses of the retina and choroid. The method has been demonstrated in the assessment of various vascular diseases such as venous occlusions, diabetic retinopathy, macular neovascularization, and others. Limitations of this imaging modality include a small registered field of view and the inability to visualize leakage and dye transit over time. It is also subject to a variety of artifacts, including those generated by blinking and eye movement during image acquisition. However, more than an alternative for FA and ICGA, OCTA is bringing new insights to our understanding of retinal and choroidal vascular structure and is changing fundamental paradigms in the clinical management of pathologic conditions. [Ophthalmic Surg Lasers Imaging Retina. 2016;47:848-861.].

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.

A review of optical coherence tomography angiography (OCTA).

Optical coherence tomography angiography (OCTA) is a new, non-invasive imaging technique that generates volumetric angiography images in a matter of seconds. This is a nascent technology with a potential wide applicability for retinal vascular disease. At present, level 1 evidence of the technology's clinical applications doesn't exist. In this paper, we introduce the technology, review the available English language publications regarding OCTA, and compare it with the current angiographic gold standards, fluorescein angiography (FA) and indocyanine green angiography (ICGA). Finally we summarize its potential application to retinal vascular diseases. OCTA is quick and non-invasive, and provides volumetric data with the clinical capability of specifically localizing and delineating pathology along with the ability to show both structural and blood flow information in tandem. Its current limitations include a relatively small field of view, inability to show leakage, and proclivity for image artifact due to patient movement/blinking. Published studies hint at OCTA's potential efficacy in the evaluation of common ophthalmologic diseases such age related macular degeneration (AMD), diabetic retinopathy, artery and vein occlusions, and glaucoma. OCTA can detect changes in choroidal blood vessel flow and can elucidate the presence of choroidal neovascularization (CNV) in a variety of conditions but especially in AMD. It provides a highly detailed view of the retinal vasculature, which allows for accurate delineation of the foveal avascular zone (FAZ) in diabetic eyes and detection of subtle microvascular abnormalities in diabetic and vascular occlusive eyes. Optic disc perfusion in glaucomatous eyes is notable as well on OCTA. Further studies are needed to more definitively determine OCTA's utility in the clinical setting and to establish if this technology may offer a non-invasive option of visualizing the retinal vasculature in detail.

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.

Stromal niche controls the plasticity of limbal and corneal epithelial differentiation in a rabbit model of recombined tissue.

PURPOSE: The adult rabbit limbal basal epithelium contains corneal epithelial stem cells, which have been characterized by a negative expression of keratin-3 (K3) and a lower expression of connexin 43 (Cx43). This study was conducted to determine whether the limbal stroma dictates the plasticity of limbal and corneal epithelial differentiation. METHODS: Viable epithelial sheets of the central cornea and the pigmented limbus were isolated from Dutch belted rabbits by incubation of 50 mg/mL of dispase II in supplemental hormonal epithelial medium (SHEM) for 18 hours at 4 degrees C. The cleavage plane was studied by immunostaining with antibodies against K3, Cx43, integrin beta1, and collagen IV. Viability of single cells derived from these sheets was assessed by a live-dead assay. Such limbal (L) and corneal (K) epithelial sheets were recombined with either limbal (Ls) or corneal (Ks) stroma, and cultured in SHEM for 10 days before lifting to the air-fluid interface for 1 week. The resultant epithelial phenotype was determined by histology and immunostaining to K3 and Cx43, and apoptosis was investigated by Hoechst and TUNEL nuclear staining. RESULTS: Viability of isolated limbal and corneal epithelial sheets was determined to be 91.1% +/- 2.9%. The basal epithelium of isolated limbal epithelial sheets was positive for integrin beta1, negative for K3, but weakly positive for Cx43, and still retained patches of collagen IV. All recombinants showed stratified epithelia, with intraepithelial cysts with desquamated debris noted only in K/Ks, and epithelial outgrowth onto the insert filter from L/Ls. As expected, expression of K3 was negative in the basal layer of L/Ls, but positive in that of K/Ks. Expression of K3 was sporadically positive in the basal layer of L/Ks but largely negative in that of K/Ls. Expression of Cx43 was uniformly expressed in the basal layer of the K/Ks, but weak in that of L/Ls, K/Ls, and L/Ks. A higher apoptosis index was only noted in intraepithelial cysts of K/Ks. CONCLUSIONS: These results strongly indicate that the limbal stroma modulates epithelial differentiation, proliferation and apoptosis in the direction favoring stemness, whereas the corneal stroma promotes differentiation. Further investigation into elements constituting such a niche should help unveil the secrecy whereby stemness is controlled.

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.

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.