Infectious Keratitis: Characterization of Microbial Diversity through Species Richness and Shannon Diversity Index.

Purpose: To characterize microbial keratitis diversity utilizing species richness and Shannon Diversity Index. Methods: Corneal impression membrane was used to collect samples. All swabs were processed and analyzed by Biolab Laboratory (level V-SSN Excellence: ISO 9001:2015), Biolab Srl (Ascoli Piceno, Italy). DNA extraction, library preparation, and sequencing were performed in all samples. After sequencing, low-quality and polyclonal sequences were filtered out by the Ion software. At this point, we employed Kraken2 for microbial community analysis in keratitis samples. Nuclease-free water and all the reagents included in the experiment were used as a negative control. The primary outcome was the reduction in bacterial DNA (microbial load) at T1, expressed as a percentage of the baseline value (T0). Richness and Shannon alpha diversity metrics, along with Bray-Curtis beta diversity values, were calculated using the phyloseq package in R. Principal coordinate analysis was also conducted to interpret these metrics. Results: 19 samples were included in the study. The results exhibited a motley species richness, with the highest recorded value surpassing 800 species. Most of the samples displayed richness values ranging broadly from under 200 to around 600, indicating considerable variability in species count among the keratitis samples. Conclusions: A significant presence of both typical and atypical bacterial phyla in keratitis infections, underlining the complexity of the disease's microbial etiology.

Clinical Outcomes of Topical 0.1% Ciclosporin Cationic Emulsion Used on Label in Children with Vernal Keratoconjunctivitis.

INTRODUCTION: The purpose of this short article is to report the clinical outcomes of topical 0.1% ciclosporin cationic emulsion (CsA-CE) used on label in children with vernal keratoconjunctivitis (VKC). METHODS: In this prospective, non-comparative, observational study children affected by active severe VKC were treated for at least 12 months with topical 0.1% CsA-CE. The drug was instilled in both eyes 4 times daily. Data collected from medical charts for the baseline visit (T0) and 1-year follow-up visit (T1) included symptomatic score (0-15), clinical score (0-15), side effects, rescue therapy (need and total number of courses with 0.1% dexamethasone 4 times daily for 5 days), ocular complications and tolerability (visual analog scale [0-100]). RESULTS: Data from 25 children (20 boys, 5 girls; mean [± standard deviation] age 8.40 ± 2.54 years) were included in the study. Of the 25 patients, 23 (92%) used 0.1% CsA-CE eye drops as per label recommendations, including four patients who had prematurely stopped using topical galenic CsA due to side effects. Symptomatic and clinical scores decreased significantly after treatment, with the mean symptomatic score decreasing from 9.76 ± 1.27 at T0 to 3.80 ± 1.08 at T1, and the mean clinical score decreasing from 9.20 ± 1.32 at T0 to 3.44 ± 1.00 at T1; both P < 0.0001). Five patients (20%) required at least one course of rescue medication (mean 3.4 ± 4.8 courses/year). No patients experienced ocular complications during the study, and treatment tolerability was very high (mean score 89.40 ± 5.46). CONCLUSION: Our findings confirm that topical CsA-CE is an effective on-label option for children with VKC in the real-life setting. In our pediatric patient population, CsA-CE provided good clinical outcomes with a limited need for rescue medication, and it was well tolerated by almost all patients, including those who were intolerant to galenic formulations.

Shotgun metagenomic sequencing in culture negative microbial keratitis.

PURPOSE: To evaluate the microbiota of culture negative Corneal Impression Membrane (CIM) microbial keratitis samples with the use of shotgun metagenomics analysis. METHODS: DNA of microbial keratitis samples were collected with CIM and extracted using the MasterPure™ Complete DNA and RNA Purification Kit (Epicentre). DNA was fragmented by sonication into fragments of 300 to 400 base pairs (bp) using Bioruptor® (Diagenode, Belgium) and then used as a template for library preparation. DNA libraries were sequenced on Illumina® HiSeq2500. The resulting reads were quality controlled, trimmed and mapped against the human reference genome. The unmapped reads were taxonomically classified using the Kraken software. RESULTS: 18 microbial keratitis samples were included in the study. Brevundimonas diminuta was found in 5 samples while 6 samples showed the presence of viral infections. Cutibacterium acnes, Staphylococcus aureus, Moraxella lacunata and Pseudomonas alcaligenes were also identified as the presumed putative cause of the infection in 7 samples. CONCLUSIONS: Shotgun sequencing can be used as a diagnostic tool in microbial keratitis samples. This diagnostic method expands the available tests to diagnose eye infections and could be clinically significant in culture negative samples.

Reoperations for complications within 90 days after gel stent implantation or trabeculectomy.

PURPOSE: To describe reoperations in the operating room for complications that occurred within the first 90 days after gel stent implantation or trabeculectomy at a single institution over 5 years. METHODS: In this retrospective chart review, patients who have undergone gel stent implantation with mitomycin C (MMC) or trabeculectomy with MMC were enrolled. Postoperative complications that required reoperations within the first 90 days were evaluated. RESULTS: A total of 510 surgeries were performed on 392 patients over a 57-month period by 2 glaucoma surgeons. Of these, 284 were gel stent implantation, and 226 were trabeculectomy. Combined phacoemulsification was performed in 52/284 (18.3%) in the gel stent group and in 26/226 (11.5%) of eyes in the trabeculectomy group (p = 0.03). Reoperations took place in 13/510 (2.5%) eyes, including 4/284 (1.4%) in the gel stent group, 9/226 (4.0%) in the trabeculectomy group (p = 0.07). In the gel stent group, indications for reoperation were bleb failure (2), suprachoroidal hemorrhage (1), bullous keratopathy (1). In the trabeculectomy group, indications for reoperation were bleb failure (3), overfiltration (2), persistent wound leak (2), aqueous misdirection (2). CONCLUSIONS: The rates of reoperation for early postoperative complications after gel stent or trabeculectomy was low and comparable with previous studies. A slightly higher number of reoperations within 90 days was observed in the trabeculectomy group than the gel stent group despite the more significant number of combined procedures in the latter group. Bleb failure was the most common indications for reoperation in both groups. Excessive outflow was a cause of reoperation mostly in the trabeculectomy group.

Nail-Patella Syndrome and Glaucoma: A Case Report and Review of the Literature.

Nail-patella syndrome (NPS) is a rare autosomal dominant disease characterized by nail dysplasia, aplastic or hypoplastic patellae, elbow dysplasia, and presence of iliac horns. Renal or ocular abnormalities are also associated with the disease. We report the case of a 57-year-old woman affected by NPS and having haploinsufficiency of the LMX1B gene who experienced severe bilateral chronic angle-closure glaucoma in both eyes and that was successfully managed with a flap-express procedure in the right eye. The left eye had no light perception, and medical treatment was considered. Glaucoma is the most frequent ocular abnormalities observed in association with NPS and usually presents with an open angle. Glaucoma associated with NPS typically has an early onset open-angle phenotype. In fewer cases, it may present with an angle-closure phenotype. Therefore, we emphasize the need for glaucoma case-finding protocols comprehensive of gonioscopy in NPS patients and their relatives.

Moving beyond the Slit-Lamp Gonioscopy: Challenges and Future Opportunities.

After almost a century from its introduction in clinical practice, slit-lamp gonioscopy is still considered the reference standard for evaluating the anterior chamber angle (ACA). Gonioscopy is essential for diagnosing angle closure disease, and ACA features are included in glaucoma's diagnostics and treatments algorithms. However, shortcomings of slit-lamp gonioscopy include a steep learning curve, lack of agreement between examiners and poor documentation. Thanks to advances in miniaturization and computing, new instruments for digital gonioscopy have been developed and marketed. This narrative review focuses on the Gonioscope GS-1, which permits semi-automated circumferential documentation of the ACA in real-colour photographs. Advantages and disadvantages of GS-1 compared with slit-lamp gonioscopy and other ACA imaging technologies such as optical coherence tomography are discussed. Finally, potential opportunities offered by this device for telemedicine, virtual clinics, and automatic classification with deep learning are presented.

Epithelial Flap Corneal Cross-linking.

PURPOSE: To compare standard epithelium-off corneal cross-linking (standard Epi-Off CXL) to corneal cross-linking with an epithelial flap (Epi-Flap CXL). METHODS: Patients who had undergone sequential bilateral CXL for progressive keratoconus were included in this comparative interventional case series. One eye was treated with the Epi-Off CXL technique and the fellow eye with the Epi-Flap CXL technique. Postoperative pain was measured using the Verbal Rating Scale and corneal densitometry using a Scheimpflug camera. Sex, age, corrected distance visual acuity, keratometry, and corneal thickness were also recorded. RESULTS: Twenty-four eyes of 12 patients with keratoconus with a mean age of 27.15 ± 5.15 years were included. The Verbal Rating Scale scores were significantly lower in patients who had Epi-Flap CXL on the first (1.00 [interquartile range (IQR): 0.00 to 1.00] vs 3.00 [IQR: 3.00 to 3.75], P = .01) and third (0.00 [IQR: 0.00 to 1.00] vs 1.00 [IQR: 0.00 to 1.00], P = .01) postoperative day compared to the Epi-Off CXL group. No pain was observed in both groups after the third day. After 12 months, the Epi-Flap CXL group showed significantly less anterior corneal haze (measured as corneal densitometry) compared to the patients treated with Epi-Off CXL (P = .01). Both groups demonstrated stability of keratometry and corneal thickness at 12 months after CXL (P < .01). CONCLUSIONS: Epi-Flap CXL is associated with less postoperative pain and anterior stromal haze in patients undergoing CXL for progressive keratoconus with no loss of efficacy. [J Refract Surg. 2021;37(11):741-745.].

Delayed Re-epithelialization After Epithelium-Off Crosslinking: Predictors and Impact on Keratoconus Progression.

Purpose: To investigate the demographic and corneal factors associated with the occurrence of delayed reepithelialization (DRE) after epithelium-off crosslinking (epi-off CXL). Design:Retrospective case series. Methods:A chart review was performed to identify patients treated with epi-off CXL. DRE was defined as a corneal epithelial defect detected by fluorescein staining that persisted for more than 10 days. Slit-lamp examination, anterior segment optical coherence tomography, corneal topography, and corneal in vivo confocal microscopy (IVCM) were always performed preoperatively and at each follow-up visit (1, 3, 6, 12 months). A generalized estimating equation was used to assess the baseline factors associated with DRE. Results:Data from 153 eyes were analyzed. The mean age of patients was 24.9 ± 8.5 years, and 47 (30.7%) were women. The average reepithelization time was 4.7 ± 1.8 days. Six eyes (3.9%) experienced DRE. In the multivariate model, both the age of the patient (OR = 1.30; p = 0.02) and the corneal steepest meridian (OR = 0.44, p = 0.047) were associated with DRE. Baseline nerve count was also associated with DRE (0.87, p = 0.03). Male gender was associated with a slower early nerve regrowth (1-6 months) (p = 0.048), but not with the occurrence of DRE (p = 0.27). Preoperative central corneal thickness was not related to DRE (p = 0.16). DRE was not associated with keratoconus progression after epi-off CXL (p = 0.520). Conclusions:The association between DRE and age may reflect the age-related decrease in the corneal healing response. Also, low baseline corneal nerve count is associated with DRE. Gender seems to affect reinnervation measured by IVCM but not the reepithelization time. DRE does not seem to affect the efficacy of epi-off CXL.

A New Surgical Technique to Deliver Riboflavin Beneath Corneal Epithelium: The Corneal Cross-Linking Epi-Pocket.

PURPOSE: To describe a new technique to deliver riboflavin into the corneal stroma during Corneal Cross-Linking (CXL) without the removal of corneal epithelium. METHODS: Keratoconus patients underwent CXL for progressive keratoconus. Riboflavin was delivered by manually creating an epithelial pocket (CXL Epi-Pocket). Verbal rating scale was recorded postoperatively. Best-corrected visual acuity, keratometric indices, corneal thickness and corneal densitometry were recorded at baseline and at 12-month follow-up. RESULTS: Eighteen eyes of 18 patients were included in the study. At a 12-month follow-up, best-corrected visual acuity, K1, K2 and densitometry values were stable. Maximum keratometry (Kmax) reduced from 55.31 ± 6.21 (SD) to 52.34d ± 4.12d (SD) (P value = 0.032). the thinnest point went from 441 ± 21.18 (SD) to 425.4 ±â€Š19.02 (SD) um (P value = 0.041). The verbal rating scale at 1, 2 and 3 days postoperatively were 1.76 ±â€Š0.19 (SD), 1.02 ±â€Š0.51 (SD) and 0.28 ±â€Š0.14 (SD). CONCLUSIONS: CXL Epi-Pocket is able to deliver riboflavin to halt the progression of keratoconus at a 12-month follow-up.

Tips, Tricks, and Guides in Descemet Membrane Endothelial Keratoplasty Learning Curve.

Lamellar keratoplasty is fast becoming the most popular form of corneal transplantation. The adoption of Descemet membrane endothelial keratoplasty (DMEK) in the management of Fuchs endothelial dystrophy and pseudophakic bullous keratopathy is partly responsible for this shift in the paradigm of management of corneal pathology. The learning curve of DMEK, however, has been proven to be much steeper than previous endothelial keratoplasty procedures. To ease the procedure, experts have proposed multiple innovative techniques from tissue preparation to graft unfolding to aid the more novice surgeon. Here, we collate and share tips and tricks from our collective experiences to support the learning curve and outcomes in DMEK for both the novice and more experienced corneal transplant surgeons.

Taking care of life: the new Italian law on post-mortem donation for study purposes, training and scientific research.

Anatomical dissection is considered a fundamental practice in the medical curriculum. Not only does it help students to familiarize themselves with the various bodily organs and to refine their surgical practice, it also fosters an attitude of awareness and respect towards death and the values of solidarity. On 10th February 2020, the Italian Parliament passed a new law entitled "Rules on the disposition of one's own body and post-mortem tissues for study purposes, training and scientific research". Here, we outline the main ethical and legal aspects of this law and suggest initiatives to encourage body donation. Through careful analysis of the law, we intend to open a discussion on the main ethical issues and to make comparisons with the international situation. The new legislation fills serious gaps in an area of great ethical relevance; not only does it endorse fundamental ethical principles, it also provides important guidelines for strengthening body donation, such as: ensuring respect for the dignity of the deceased, providing express consent, appointing a trustee, promoting information for both doctors and the community, and banning all forms of trade in bodies or body parts. This law is certainly an important milestone on the road to overcoming the scarcity of bodies for study and research. However, it is also essential to promote a favourable attitude to body donation and to strive to overcome obstacles to this practice by supporting continuous dialogue between anatomical institutes and the community. Further studies are necessary in order to assess the impact of the new law.

Less is more: new one-step intracameral chemotherapy technique.

Purpose: To describe the feasibility of a new one-step approach to aspirate the aqueous and apply melphalan in a single-go without repeated entries into the anterior chamber. Methods: This retrospective non-comparative study was conducted at a referral center and included 12 patients. The one-step approach is described in a step-wise manner. No complications were observed among the patients. Results: One single injection of intracameral melphalan proved to be a successful treatment in nine cases. Two patients required a second injection, which was administered two weeks after the first one following the same technique. Conclusions: This proved to be a reasonable technique for the smooth application of melphalan in the anterior chamber studded with retinoblastoma seeds. Our outcomes revealed that it is an effective, quick, and cost-effective technique. Longer-term data collection is underway, though initial findings are encouraging.

Recombinant Human Nerve Growth Factor (Cenegermin)-Driven Corneal Wound Healing Process: An Evidence-Based Analysis.

Purpose: To evaluate anterior segment optical coherence tomography (AS-OCT) to detect the wound healing process as per monitoring the effectiveness of cenegermin to treat moderate to severe neurotrophic keratoplasty. Methods: A retrospective chart review was realized to identify patients treated with cenegermin at the Clinica Oculistica, University of Genoa, Italy. All patients underwent careful examinations at baseline and follow-up visits. AS-OCT scans centered on the minimum corneal thickness (CT) area were always performed. We compared findings of AS-OCT with the findings from the slit-lamp examination. A linear regression analysis was used to evaluate factors associated with corneal healing. A further analysis, including a control group treated with 50% autologous serum (AS), was done to investigate and compare the efficacy of cenegermin. Results: Data from 16 eyes were studied. The average patients' age was 60.9 ± 21.1 years; five (31.2%) eyes experienced persistent epithelial defect and 11 (68.8%) eyes had neurotrophic corneal ulcer. The average reepithelialization time was 3.9 ± 0.5 weeks in the cenegermin group versus 5.9 ± 1.9 weeks in the AS group (p < 0.01). The AS-OCT scans revealed an average CT at the thinnest point of 276.3 ± 74.1 µm before treatment with an average increase of 176.5 ± 60.3 µm at the end of the cenegermin treatment (B = -0.15; p = 0.035). The AS-OCT percentage increase in corneal thickness between the two groups was statistically significant (p < 0.02). Conclusion: Understanding the cascade of events involved in the nerve growth factor-driven corneal wound healing process is clinically meaningful for the clinician. AS-OCT is an effective tool for systematic anterior segment imaging, allowing the detailed detection of the front-to-back layered corneal structure for quantitative analysis and monitoring of the healing process.

Plug the pit: a surgical technique for optic disc pit.

Purpose: To present a recently described surgical technique for the treatment of optic disc pit (ODP) and evaluate its outcomes. Methods: A patient presented with refractory serous macular detachment and secondary full thickness macular hole associated with ODP, for which he had already undergone pars-plana vitrectomy with internal limiting membrane peeling and autologous serum application over the optic disc pit. A recently described surgical technique was carried out to treat this case. In this procedure, a silicone punctal plug was used to close the ODP. The macular hole was closed with a human amniotic membrane graft. Endotamponade was carried out with 1000cs silicone oil. Results: Postoperatively, the serous macular detachment subsided and the punctal plug and amniotic membrane graft were in situ. Patient's visual acuity improved from counting fingers to 6/38 at one year postoperative. Conclusion: This technique appears to be safe and effective in resolving long standing serous macular detachment associated with ODP, which was refractory to the conventional intervention. However, more cases and longer follow-ups are needed to affirm the safety and efficacy of this recently described procedure.

Solar retinopathy: a new setting of red, green, and blue channels.

PURPOSE: To introduce a new color imaging technique using improved settings of red, green, and blue channels for improved delineation of retinal damage in patients with solar retinopathy. METHOD: A retrospective case series of patients with poor vision secondary to solar retinopathy were analyzed. All patients underwent visual acuity, refraction, and dilated fundus examination. A spectral domain-optical coherence tomography of the macula and color fundus imaging using optimized red, green, and blue color setting was performed. Patients were reviewed over a 6-month period. The data were analyzed for statistical significance using an independent t test and a receiver operating characteristic curve. RESULTS: In total, 20 eyes of 10 patients were included between 2009 and 2017. The mean age was 24.9 ± 18.1 years. Best corrected visual acuity at first consultation was 0.78 ± 0.11 and after 6 months was 0.83 ± 0.09. Spectral domain-optical coherence tomography demonstrated retinal abnormalities at the myoid zone, ellipsoid zone, and the outer segment of photoreceptors. Receiver operating characteristic curve analysis showed an improving effect (area under the curve = 0.62; 95% confidence interval = 0.42-0.79). The color channels parameters, which improve visualization of the lesions were found to be 67-0.98-255 for the R-guided setting, 19-0.63-121 for the B-guided setting, and 7-1.00-129 for the G-guided setting. The ideal red, green, and blue setting was in 24-0.82-229. CONCLUSION: The use of a new setting of red, green, and blue channels could improve the diagnosis and monitoring of solar retinopathy, hence improving patient care.

Early Postoperative Intraocular Pressure is Associated With Better Pressure Control After XEN Implantation.

PRéCIS:: Gel stent implantation is a bleb-forming surgery designed to achieve predictable pressure drop. An early low intraocular pressure (IOP) is associated with the long-term success of the procedure. PURPOSE: To identify the variables associated with the success of the XEN procedure. PATIENTS AND METHODS: This was part of a prospective, uncontrolled, consecutive case series study. Patients with primary open-angle glaucoma or pseudo-exfoliative glaucoma were included. All the patients underwent surgical XEN implant procedure with mitomycin-C subconjunctival injection 20 minutes before surgery. Success criteria were an off-medication IOP of 6 to 16 mm Hg 12 months after surgery; no additional glaucoma surgery; no visual threatening complications, no visual acuity loss >1 Snellen line. One eye per patient was considered for statistical analysis. A univariate Cox proportional hazard regression analysis was performed to identify potential risk factors for surgical failure. Then, a multivariate Cox model was built. RESULTS: One hundred twenty-three patients were recruited in this study: 93 patients underwent XEN implantation alone whereas 30 the combined procedure with phacoemulsification and intraocular lens implantation. Univariate Cox regression showed that the day after surgery intraocular lens>9 mm Hg was associated with surgical failure (P=0.02) and a postoperative number of needlings ≥2 in the follow-up was also predictive of surgical failure (P<0.01). These data were confirmed by a multivariate model too. At 1 year the surgical success criteria were 76% in the group with 24-hour IOP≤9 mm Hg, whereas it was 43% when above 9 mm Hg (P=0.026). CONCLUSIONS: Our study shows that an early IOP≤9 mm Hg is predictive of the efficacy of the procedure during 1-year follow-up, whereas more than 2 needlings are predictive of failure.

CFSE: A New Method for Identifying Human Limbal Stem Cells and Following Their Migration in Human Cornea.

AIM: To develop a method capable of identifying human corneal limbal stem cells (LSCs) and follow their proliferation and migration in the epithelium. MATERIALS AND METHODS: Ten fresh matched pairs of cadaveric normal human corneas were obtained from donors. Carboxyfluorescein diacetate succinimidyl ester (CFSE) was used to target LSCs. The distribution of CFSE-positive cell clusters was analyzed by fluorescence microscopy by counterstaining with 4',6-diamidino-2-phenylindole (DAPI). Fluorescence was digitally recorded for seven days, and the rate of cell movement was determined. RESULTS: CFSE-labeled cells were tracked in corneas. Analysis of time sequences revealed that they moved centripetally. Daily average CFSE-labeled LSC movement was 0.073±0.01 cm (±SD). CONCLUSION: CFSE allowed us to identify LSCs and to track their centripetal migration from the limbal basal layer to the anterior ocular surface. This experimental system appears to be a valuable tool for further studies on corneal epithelial cell migration and proliferation.

Predictors of Endothelial Cell Loss after Phacoemulsification for the Treatment of Primary Angle Closure.

PURPOSE: To investigate demographic and anatomical factors associated with a reduction in endothelial cell density (ECD) after phacoemulsification (PE) for the treatment of primary angle closure (PAC). METHODS: In this prospective case series, ECD was evaluated by noncontact specular microscopy and biometric parameters by both noncontact optical biometry and anterior segment optical coherence tomography, preoperatively and at 12 months after surgery. Anterior segment biomicroscopy and gonioscopy were also performed. The change in ECD and its relation to clinical characteristics and biometric parameters were evaluated by linear regression analysis. RESULTS: 44 patients with PAC were included in the study. The mean (SD) patient age was 71.6 (10.2) years; thirty-one (70.5%) of them were women. Coexistence of exfoliation syndrome (XS) was observed in 4 cases (9.1%). The mean (SD) ECD (cells/mm2) changed from 2275 (463) preoperatively to 1964 (613) postoperatively with a mean reduction of -310 (95% CI -445 to -176; p < 0.001). In the multivariate regression model, after correction for age and lens status, XS was the only parameter associated with ECD percentage change (B = -36.00; p=0.001). CONCLUSION: PE in angle closure causes a significant ECD reduction. In our population of PAC patients, XS is significantly associated with ECD change. In this group of patients, a careful preoperative endothelial evaluation should be performed.

Collagen Cross-Linking in the Management of Microbial Keratitis.

Increasing resistance to antimicrobial agents has contributed to an elevated risk of complications of infectious keratitis. Corneal collagen cross-linking (CXL) has been widely adopted for the management of keratoconus and post-refractive surgery corneal ectasia. It has recently been introduced as an option for treating keratitis due to multidrug resistant organisms. The purpose of this review is to discuss the rationale, safety, and evidence for CXL in infectious keratitis and its possible effect on ocular surface inflammation. Published data show that CXL is effective and safe as an adjunct to antibiotic treatment in selected cases of bacterial keratitis. The benefit of CXL probably varies according to the etiology of the infection.