Association between Moraxella keratitis and advanced glycation end products.

Diabetes mellitus is recognized as a major predisposing factor for Moraxella keratitis. However, how diabetes mellitus contributes to Moraxella keratitis remains unclear. In this study, we examined Moraxella keratitis; based on the findings, we investigated the impact of advanced glycation end products (AGEs) deposition in the cornea of individuals with diabetic mellitus on the adhesion of Moraxella isolates to the cornea. A retrospective analysis of 27 culture-proven cases of Moraxella keratitis at Ehime University Hospital (March 2006 to February 2022) was performed. Moraxella isolates were identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Among the patients, 30.4% had diabetes mellitus and 22.2% had the predominant ocular condition of using steroid eye drops. The species identified were Moraxella nonliquefaciens in 59.3% and Moraxella lacunata in 40.7% of patients. To investigate the underlying mechanisms, we assessed the effects of M. nonliquefaciens adherence to simian virus 40-immortalized human corneal epithelial cells (HCECs) with or without AGEs. The results demonstrated the number of M. nonliquefaciens adhering to HCECs was significantly increased by adding AGEs compared with that in controls (p < 0.01). Furthermore, in the corneas of streptozotocin-induced diabetic C57BL/6 mice treated with or without pyridoxamine, an AGE inhibitor, the number of M. nonliquefaciens adhering to the corneas of diabetic mice was significantly reduced by pyridoxamine treatment (p < 0.05). In conclusion, the development of Moraxella keratitis may be significantly influenced by the deposition of AGEs on the corneal epithelium of patients with diabetes mellitus.

Insulin eye drops improve corneal wound healing in STZ-induced diabetic mice by regulating corneal inflammation and neuropeptide release.

INTRODUCTION: In recent years, insulin eye drops have attracted increasing attention from researchers and ophthalmologists. The aim of this study was to investigate the efficacy and possible mechanism of action of insulin eye drops in diabetic mice with corneal wounds. METHODS: A type 1 diabetes model was induced, and a corneal epithelial injury model of 2.5 mm was established. We used corneal fluorescein staining, hematoxylin-eosin (H-E) staining and the Cochet-Bonnet esthesiometer to examine the process of wound healing. Subsequently, the expression levels of Ki-67, IL-1ß, ß3-tubulin and neuropeptides, including substance P (SP) and calcitonin gene-related peptide (CGRP), were examined at 72 h after corneal injury. RESULTS: Fluorescein staining demonstrated an acceleration of the recovery of corneal epithelial injury in diabetic mice compared with the saline treatment, which was further evidenced by the overexpression of Ki-67. Moreover, 72 h of insulin application attenuated the expression of inflammatory cytokines and neutrophil infiltration. Remarkably, the results demonstrated that topical insulin treatment enhanced the density of corneal epithelial nerves, as well as neuropeptide SP and CGRP release, in the healing cornea via immunofluorescence staining. CONCLUSIONS: Our results indicated that insulin eye drops may accelerate corneal wound healing and decrease inflammatory responses in diabetic mice by promoting nerve regeneration and increasing levels of neuropeptides SP and CGRP.

Glycol Chitosan-Poly(lactic acid) Conjugate Nanoparticles Encapsulating Ciprofloxacin: A Mucoadhesive, Antiquorum-Sensing, and Biofilm-Disrupting Treatment Modality for Bacterial Keratitis.

Bacterial keratitis (BK) causes visual morbidity/blindness if not treated effectively. Here, ciprofloxacin (CIP)-loaded nanoparticles (NPs) using glycol chitosan (GC) and poly(lactic acid) (PLA) conjugate at three different ratios (CIP@GC(PLA) NPs (1:1,5,15)) were fabricated. CIP@GC(PLA) NPs (1:1) were more effective than other tested ratios, indicating the importance of optimal hydrophobic/hydrophilic balance for corneal penetration and preventing bacterial invasion. The CIP@GC(PLA) (NPs) (1:1) realized the highest association with human corneal epithelial cells, which were nonirritant to the hen's egg-chorioallantoic membrane test (HET-CAM test) and demonstrated significant antibacterial response in the in vitro minimum inhibitory, bactericidal, live-dead cells, zone of inhibition, and biofilm inhibition assays against the keratitis-inducing pathogen Pseudomonas aeruginosa. The antiquorum sensing activity of GC has been explored for the first time. The NPs disrupted the bacterial quorum sensing by inhibiting the production of virulence factors, including acyl homoserine lactones, pyocyanin, and motility, and caused significant downregulation of quorum sensing associated genes. In the in vivo studies, CIP@GC(PLA) NPs (1:1) displayed ocular retention in vivo (∼6 h) and decreased the opacity and the bacterial load effectively. Overall, the CIP@GC(PLA) NP (1:1) is a biofilm-disrupting antiquorum sensing treatment regimen with clinical translation potential in BK.

Five-Year Risk and Safety Profile of Autologous Serum Eye Drop Therapy.

PURPOSE: To evaluate the risk and safety profile of autologous serum eye drop therapy in clinical routine over a period of 5 years. METHODS: This retrospective study involved all patients treated with autologous serum between July 2014 and December 2019 at a tertiary ophthalmic referral center. The electronic patient record system was searched for all patients with autologous serum eye drop therapy at any time point. These records were subsequently searched for keywords such as infectious keratitis, corneal ulcer, conjunctivitis, or endophthalmitis at any recorded contact. The probability of an association between the therapy with autologous serum eye drops and infectious complications was investigated independently by three corneal specialists and rated as likely, potential, or unlikely. RESULTS: In total, 752 patients were treated with autologous serum eye drops between July 2014 and December 2019. There were 5 384 batches with a total of 107 680 bottles of serum eye drops that had been produced and dispensed for these patients during this period. The records of 291 patient showed a combination of autologous serum therapy and at least one keyword for infectious diseases. In 288 patients, individual case analyses revealed an unlikely association between the therapy and infection, as their infectious episodes occurred either before the start of the therapy, more than 1 month after the therapy ceased, or in the contralateral untreated eye in the case of unilateral therapy. Three cases of infectious keratitis were classified as potentially associated with autologous serum therapy. However, all three patients suffered from chronic anterior eye diseases with a high risk of spontaneous infectious complications independent of therapy with autologous eye drops. None of the infectious events was rated as being likely due to the serum eye drops. CONCLUSIONS: Serum eye drops are often used in patients with severe or chronic anterior eye diseases with an intrinsic risk of infectious diseases. Despite these preexisting risk factors, autologous serum eye drops can be considered safe, even in patients with a compromised ocular surface.

Microbial keratitis in Southern Malawi: a microbiological pilot study.

OBJECTIVE: Microbial keratitis (MK) is a significant cause of blindness in sub-Saharan Africa. We investigated the feasibility of using a novel corneal impression membrane (CIM) for obtaining and processing samples by culture, PCR and whole-genome sequencing (WGS) in patients presenting with suspected MK in Malawi. METHODS AND ANALYSIS: Samples were collected from patients presenting with suspected MK using a 12 mm diameter polytetrafluoroethylene CIM disc. Samples were processed using culture and PCR for Acanthamoeba, herpes simplex virus type 1 (HSV-1) and the bacterial 16S rRNA gene. Minimum inhibitory concentrations of isolates to eight antimicrobials were measured using susceptibility strips. WGS was used to characterise Staphylococcus aureus isolates. RESULTS: 71 eyes of 71 patients were included. The overall CIM isolation rate was 81.7% (58 positive samples from 71 participants). 69 (81.2%) of isolates were Gram-positive cocci. Coagulase-negative Staphylococcus 31.8% and Streptococcus species 14.1% were the most isolated bacteria. Seven (9.9%) participants were positive for HSV-1. Fungi and Acanthamoeba were not detected. Moxifloxacin and chloramphenicol offered the best coverage for both Gram-positive and Gram-negative isolates when susceptibility was determined using known antimicrobial first quartile concentrations and European Committee on Antimicrobial Susceptibility Testing breakpoints, respectively. WGS identified known virulence genes associated with S. aureus keratitis. CONCLUSIONS: In a resource-poor setting, a CIM can be used to safely sample the cornea in patients presenting with suspected MK, enabling identification of causative microorganisms by culture and PCR. Although the microbiological spectrum found was limited to the dry season, these preliminary results could be used to guide empirical treatment.

Ophthalmic manifestations as the first presenting feature in dengue fever: a 10-year study.

Purpose: To report patients who first presented with various ocular manifestations and eventually ascertained to have underlying dengue. Methods: A prospective study was conducted at multiple tertiary eye-care centers in India from 2012 to 2022. Cases reporting initially with ocular features along with fever/past history of fever over the last two weeks or with clinical features of dengue were selected. After an ophthalmological examination, patients underwent complete serological and biochemical analysis and those with reduced platelet counts were evaluated for dengue. Results: Out of 564 cases, 15 patients were verified to be afflicted with dengue eventually. A rising trend of cases was seen every year and out of 15 cases, eight cases were reported during the Covid-19 pandemic (from 2020 to 2022), but were COVID-negative. 9 cases presented with acute redness followed by diminished vision. Seven cases presented a history of fever over the last few days and one had traveled from dengue endemic area. The various ocular presentations included subconjunctival hemorrhage, viral keratitis, anterior uveitis, sixth-nerve palsy, and vitreous hemorrhage. On serological examination, all 15 patients were detected to have low platelets. All cases responded well with supportive treatment and the ocular features subsided in all within a couple of weeks with good visual recovery. Conclusion: In a tropical nation, such as India, with endemic dengue zones and increasing figures of dengue lately, ophthalmologists must include dengue fever among the differential diagnoses in various ocular presentations like subconjunctival hemorrhage, viral keratitis, anterior uveitis, sixth nerve palsy, and vitreous hemorrhage. Abbreviations: DHF = dengue hemorrhagic fever, PCR = polymerase chain reaction, RT-PCR = real-time automated reverse transcriptase (RT-PCR), SD = standard deviation, MAC-ELIS = IgM antibodies capture enzyme-linked immunosorbent assay, RE = right eye, LE = left eye, CECT = Contrast-enhanced computed tomography.

Whole Genome Sequencing Highlights the Pathogenic Profile in Nocardia Keratitis.

Purpose: Nocardia keratitis is a serious and sight-threatening condition. This study aims to reveal the virulence and antimicrobial resistance gene profile of Nocardia strains using whole genome sequencing. Methods: Whole-genome sequencing was performed on 23 cornea-derived Nocardia strains. Together with genomic data from the respiratory tract and the environment, 141 genomes were then utilized for phylogenetic and pan-genome analyses, followed by virulence and antibiotic resistance analysis. The correlations between virulence genes and pathogenicity were experimentally validated, including the characteristics of Nocardia colonies and clinical and histopathological evaluations of Nocardia keratitis mice models. Results: Whole-genome sequencing of 141 Nocardia strains revealed a mean of 220 virulence genes contributed to bacterial pathogenesis. The mce gene family analysis led to the categorization of strains from the cornea into groups A, B, and C. The colonies of group C had the largest diameter, height, and fastest growth rate. The size of corneal ulcers and the clinical scores showed a significant increase in mouse models induced by group C. The relative expression levels of pro-inflammatory cytokines (CD4, IFN-γ, IL-6Rα, and TNF-α) in the lesion area exhibited an increasing trend from group A to group C. Antibiotic resistance genes (ARGs) spanned nine distinct drug classes, four resistance mechanisms, and seven primary antimicrobial resistance gene families. Conclusions: Whole genome sequencing highlights the pathogenic role of mce gene family in Nocardia keratitis. Its distribution pattern may contribute to the distinct characteristics of the growth of Nocardia colonies and the clinical severity of the mice models.

Spectrum and antibiotic sensitivity of bacterial keratitis: a retrospective analysis of eight years in a Tertiary Referral Hospital in Southwest China.

Purpose: The objective of this study was to investigate the epidemiological characteristics, distribution of isolates, prevailing patterns, and antibiotic susceptibility of bacterial keratitis (BK) in a Tertiary Referral Hospital located in Southwest China. Methods: A retrospective analysis was conducted on 660 cases of bacterial keratitis occurring between January 2015 and December 2022. The demographic data, predisposing factors, microbial findings, and antibiotic sensitivity profiles were examined. Results: Corneal trauma emerged as the most prevalent predisposing factor, accounting for 37.1% of cases. Among these cases, bacterial culture results were positive in 318 cases, 68 species of bacteria were identified. The most common Gram-Positive bacteria isolated overall was the staphylococcus epidermis and the most common Gram-Negative bacteria isolated was Pseudomonas aeruginosa. Methicillin-Resistant Staphylococci accounted for 18.1% of all Gram-Positive bacteria. The detection rate of P. aeruginosa showed an increasing trend over time (Rs=0.738, P=0.037). There was a significant decrease in the percentage of Gram-Negative microorganisms over time (Rs=0.743, P=0.035). The sensitivity of Gram-Positive bacteria to linezolid, vancomycin, tigecycline, quinupristin/dalfopristin, and rifampicin was over 98%. The sensitivity rates of Gram-Negative bacteria to amikacin, meropenem, piperacillin/tazobactam, cefoperazone sodium/sulbactam, ceftazidime, and cefepime were all above 85%. In patients with a history of vegetative trauma, the possibility of BK should be taken into account in addition to the focus on fungal keratitis. Conclusion: The microbial composition primarily consists of Gram-Positive cocci and Gram-Negative bacilli. Among the Gram-Positive bacteria, S. epidermidis and Streptococcus pneumoniae are the most frequently encountered, while P. aeruginosa is the predominant Gram-Negative bacteria. To combat Gram-Positive bacteria, vancomycin, linezolid, and rifampicin are considered excellent antimicrobial agents. When targeting Gram-Negative pathogens, third-generation cephalosporins exhibit superior sensitivity compared to first and second-generation counterparts. As an initial empirical treatment for severe cases of bacterial keratitis and those unresponsive to fourth-generation fluoroquinolones in community settings, the combination therapy of vancomycin and tobramycin is a justifiable approach. Bacterial keratitis can be better managed by understanding the local etiology and antibacterial drug susceptibility patterns.

Cathelicidin boosts the antifungal activity of neutrophils and improves prognosis during Aspergillus fumigatus keratitis.

Aspergillus fumigatus (A. fumigatus) is one of the common pathogens of fungal keratitis. Fungal growth and invasion cause excessive inflammation and corneal damage, leading to severe vision loss. Neutrophils are the primary infiltrating cells critical for fungal clearance. Cathelicidin [LL-37 in humans and cathelicidin-related antimicrobial peptide (CRAMP) in mice], a natural antimicrobial peptide, can directly inhibit the growth of many pathogens and regulate immune responses. However, the role of cathelicidin and its effect on neutrophils in A. fumigatus keratitis remain unclear. By establishing A. fumigatus keratitis mouse models, we found that cathelicidin was increased in A. fumigatus keratitis. It could reduce fungal loads, lower clinical scores, and improve corneal transparency. Restriction of CRAMP on fungal proliferation was largely counteracted in CD18-/- mice, in which neutrophils cannot migrate into infected sites. When WT neutrophils were transferred into CD18-/- mice, corneal fungal loads were distinctly reduced, indicating that neutrophils are vital for CRAMP-mediated resistance. Furthermore, cathelicidin promoted neutrophils to phagocytose and degrade conidia both in vitro and in vivo. CXC chemokine receptor 2 (CXCR2) was reported to be a functional receptor of LL-37 on neutrophils. CXCR2 antagonist SB225002 or phospholipase C (PLC) inhibitor U73122 weakened LL-37-induced phagocytosis. Meanwhile, LL-37 induced PLC γ phosphorylation, which was attenuated by SB225002. SB225002 or the autophagy inhibitors Bafilomycin-A1 and 3-Methyladenine weakened LL-37-induced degradation of conidia. Transmission electron microscopy (TEM) observed that LL-37 increased autophagosomes in Aspergillus-infected neutrophils. Consistently, LL-37 elevated autophagy-associated protein expressions (Beclin-1 and LC3-II), but this effect was weakened by SB225002. Collectively, cathelicidin reduces fungal loads and improves the prognosis of A. fumigatus keratitis. Both in vitro and in vivo, cathelicidin promotes neutrophils to phagocytose and degrade conidia. LL-37/CXCR2 activates PLC γ to amplify neutrophils' phagocytosis and induces autophagy to eliminate intracellular conidia.

Mesoporous zinc oxide-based drug delivery system offers an antifungal and immunoregulatory strategy for treating keratitis.

Fungal keratitis is a refractory eye disease that is prone to causing blindness. Fungal virulence and inflammatory responses are two major factors that accelerate the course of fungal keratitis. However, the current antifungal drugs used for treatment usually possess transient residence time on the ocular surface and low bioavailability deficiencies, which limit their therapeutic efficacy. In this work, natamycin (NATA)-loaded mesoporous zinc oxide (Meso-ZnO) was synthesized for treating Aspergillus fumigatus keratitis with excellent drug-loading and sustained drug release capacities. In addition to being a carrier for drug delivery, Meso-ZnO could restrict fungal growth in a concentration-dependent manner, and the transcriptome analysis of fungal hyphae indicated that it inhibited the mycotoxin biosynthesis, oxidoreductase activity and fungal cell wall formation. Meso-ZnO also promoted cell migration and exhibited anti-inflammatory role during fungal infection by promoting the activation of autophagy. In mouse models of fungal keratitis, Meso-ZnO/NATA greatly reduced corneal fungal survival, alleviated tissue inflammatory damage, and reduced neutrophils accumulation and cytokines expression. This study suggests that Meso-ZnO/NATA can be a novel and effective treatment strategy for fungal keratitis.

Biophilic Positive Carbon Dot Exerts Antifungal Activity and Augments Corneal Permeation for Fungal Keratitis.

Fungal keratitis (FK) is an infectious eye disease that poses a significant risk of blindness. However, the effectiveness of conventional antifungal drugs is limited due to the intrinsic ocular barrier that impedes drug absorption. There is an urgent need to develop new therapeutic strategies to effectively combat FK. Herein, we synthesized an ultrasmall positively charged carbon dot using a simple stage-melting method. The carbon dot can penetrate the corneal barrier by opening the tight junctions, allowing them to reach the lesion site and effectively kill the fungi. The results both in vitro and in vivo demonstrated that it exhibited good biocompatibility and antifungal activity, significantly improving the therapeutic effect in a mouse model of FK. Therefore, this biophilic ultrasmall size and positive carbon dot, characterized by its ability to penetrate the corneal barrier and its antifungal properties, may offer valuable insights into the design of effective ocular nanomedicines.

Cationized gelatin-sodium alginate polyelectrolyte nanoparticles encapsulating moxifloxacin as an eye drop to treat bacterial keratitis.

A mucoadhesive polyelectrolyte complex (PEC) nanoparticles were developed for ocular moxifloxacin (Mox) delivery in Bacterial Keratitis (BK). Moxifloxacin-loaded G/CG-Alg NPs were prepared by an amalgamation of cationic polymers (gelatin (G)/cationized gelatin (CG)), and anionic polymer (sodium alginate (Alg)) along with Mox respectively. Mox@CG-Alg NPs were characterized for physicochemical parameters such as particle size (DLS technique), morphology (SEM analysis), DSC, XRD, encapsulation efficiency, drug loading, mucoadhesive study (by texture analyzer), mucin turbidity, and viscosity assessment. The NPs uptake and toxicity of the formulation were analyzed in the Human Corneal Epithelial (HCE) cell line and an ocular irritation study was performed on the HET-CAM. The results indicated that the CG-Alg NPs, with optimal size (217.2 ± 4 nm) and polydispersity (0.22 ± 0.05), have shown high cellular uptake in monolayer and spheroids of HCE. The drug-loaded formulation displayed mucoadhesiveness, trans-corneal permeation, and sustained the release of the Mox. The anti-bacterial efficacy studied on planktonic bacteria/biofilms of P. aeruginosa and S. aureus (in vitro) indicated that the Mox@CG-Alg NPs displayed low MIC, higher zone of bacterial growth inhibition, and cell death compared to free Mox. A significant reduction of bacterial load was observed in the BK-induced mouse model.

Applications of photodynamic therapy in keratitis.

Keratitis is corneal inflammatory disease which may be caused by several reason such as an injury, allergy, as well as a microbial infection. Besides these, overexposure to ultraviolet light and unhygienic practice of contact lenses are also associated with keratitis. Based on the cause of keratitis, different lines of treatments are recommended. Photodynamic therapy is a promising approach that utilizes light activated compounds to instigate either killing or healing mechanism to treat various diseases including both communicable and non-communicable diseases. This review focuses on clinically-important patent applications and the recent literature for the use of photodynamic therapy against keratitis.

Deoxynivalenol Damages Corneal Epithelial Cells and Exacerbates Inflammatory Response in Fungal Keratitis.

BACKGROUND: Fungal keratitis (FK) is a kind of infectious keratopathy with a high rate of blindness worldwide. Deoxynivalenol (DON) has been proven to have multiple toxic effects on humans and animals. OBJECTIVES: The aim of this study was to explore a possible pathogenic role of DON in FK. METHODS: We first made an animal model of FK in New Zealand white rabbits, and then attempted to detect DON in a culture medium in which Fusarium solani had been grown and also in the corneal tissue of the animal model of Fusarium solani keratitis. Next, a model of DON damage in human corneal epithelial cells (HCECs) was constructed to evaluate effects of DON on the activity, migration ability, cell cycle, and apoptosis in the HCECs. Then, putative the toxic damaging effects of DON on rabbit corneal epithelial cells and the impact of the repair cycle were studied. The expression levels of inflammatory factors in the corneas of the animal model and in the model of DON-damaged HCECs were measured. RESULTS: The Fusarium solani strain used in this study appeared to have the potential to produce DON, since DON was detected in the corneal tissue of rabbits which had been inoculated with this Fusarium solani strain. DON was found to alter the morphology of HCECs, to reduce the activity and to inhibit the proliferation and migration of HCECs. DON also induced the apoptosis and S-phase arrest of HCECs. In addition, DON was found to damage rabbit corneal epithelial cells, to prolong the corneal epithelial regeneration cycle, and to be associated with the upregulated expression of inflammatory factors in HCECs and rabbit corneas. CONCLUSIONS: DON appears to have a toxic damaging effect on HCECs in FK, and to induce the expression of inflammatory factors, leading to the exacerbation of keratitis and the formation of new blood vessels. Future studies will explore the possibility of developing a test to detect DON in ophthalmic settings to aid the rapid diagnosis of FK, and to develop DON neutralizers and adsorbents which have the potential to improve keratocyte status, inhibit apoptosis, and alleviate inflammation, therein providing new thinking for therapy of clinical FK.

Extracellular vesicles from Candida albicans modulate immune cells function and play a protective role in fungal keratitis.

Fungal keratitis (FK) is a highly blinding infectious corneal disease caused by pathogenic fungi. Candida albicans (C. albicans) is one of the main pathogens of fungal keratitis. Extracellular vesicles (EVs), lipid bilayer compartments released by almost all living cells, including fungi, have garnered attention for their role in pathogenic microbial infection and host immune responses in recent years. Studies have reported that pretreating the host with fungal EVs can reduce the inflammatory response of the host when attacked by fungi and reduce the lethality of fungal infection. However, there are no studies that have evaluated whether C. albicans EVs can modulate the inflammatory response associated with C. albicans keratitis. Our study revealed that C. albicans EVs could activate the polymorphonuclear cells (PMNs) and promote their secretion of proinflammatory cytokines and nitric oxide (NO), enhance their phagocytic and fungicidal abilities against C. albicans. C. albicans EVs also induced a proinflammatory response in RAW264.7 cells, which was characterized by increased production of inflammatory cytokines and elevated expression of the chemokine CCL2. Similarly, stimulation of C. albicans EVs to RAW264.7 cells also enhanced the phagocytosis and killing ability of cells against C. albicans. Besides, in our in vivo experiments, after receiving subconjunctival injection of C. albicans EVs, C57BL/6 mice were infected with C. albicans. The results demonstrated that pre-exposure to C. albicans EVs could effectively diminish the severity of keratitis, reduce fungal load and improve prognosis. Overall, we conclude that C. albicans EVs can modulate the function of immune cells and play a protective role in C. albicans keratitis.

[Subfamily of betaherpesviruses as a cause of epithelial and stromal keratitis]. / Podsemeistvo beta-gerpesvirusov kak prichina epitelial'nykh i stromal'nykh keratitov.

PURPOSE: This article presents the first clinical results of intravital morphological verification of epithelial and stromal keratitis associated with betaherpesviruses. MATERIAL AND METHODS: The study group included 12 patients (12 eyes) diagnosed with herpetic keratitis. During the initial visit to the clinic, each patient underwent a standard ophthalmological examination, as well as a number of laboratory tests: immunochemical analysis of blood, molecular diagnostics, and confocal microscopy. Histological study of the cornea was additionally performed in cases indicated for surgical treatment (2 patients). RESULTS: According to enzyme-linked immunoelectrodiffusion essay (ELISA), acute-phase immunoglobulins of class M (Ig M) to cytomegalovirus (CMV) were detected in only one clinical case. Class G immunoglobulins (Ig G) to both CMV and human herpes virus type 6 (HHV-6) were detected in the majority of cases. Quantitative polymerase chain reaction (qPCR) revealed CMV DNA in tears (2 patients) and in saliva (4 patients). The HHV-6 genome was found in tears (2 patients) and in saliva (3 patients). According to the results of confocal microscopy, owl's eye cells were found in 8 patients of the group. Histological examination of the cornea helped identify pathognomonic cells in one case. Thus, 8 patients of the group were diagnosed with keratitis associated with the betaherpesvirus subfamily. CONCLUSION: Results of observation of the study patients suggest the possibility of developing keratitis associated with the subfamily of betaherpesviruses with localization in the superficial layers of the cornea. Confocal microscopy can be useful for identification of pathognomonic owl's eye cells in the corneal tissues and confirmation of the diagnosis of betaherpesvirus-associated keratitis. Investigation of the etiological factor of superficial and stromal viral keratitis is important for determining the further tactics of pharmacotherapy.

Amniotic membrane transplantation for neurotrophic corneal ulcers.

PURPOSE: To evaluate the clinical results of cryopreserved amniotic membrane transplantation as a treatment option for refractory neurotrophic corneal ulcers. METHODS: This prospective study included 11 eyes of 11 patients who underwent amniotic membrane transplantation for the treatment of refractory neurotrophic corneal ulcers at Hospital de Clínicas da Universidade Federal do Paraná, in the city of Curitiba, from May 2015 to July 2021. Patients underwent different surgical techniques in which the amniotic membrane was applied with the epithelium facing upward to promote corneal re-epithelialization. RESULTS: The median age of the patients was 60 years (range, 34-82 years), and 64% were men. The predominant etiology of corneal ulcers was herpes zoster (45% of cases). Approximately one-third of the patients (27%) were chronically using hypotensive eye drops, and more than half (54%) had previously undergone penetrating corneal transplantation. At the time of amniotic membrane transplantation, 18% of the eyes had corneal melting, 9% had corneal perforation, and the others had corneal ulceration without other associated complications (73%). The time between clinical diagnosis and surgical treatment ranged from 9 days to 2 years. The corrected visual acuity was worse than 20/400 in 90% of the patients preoperatively, with improvement in 36% after 3 months of the procedure, worsening in 18% and remaining stable in 36%. Of the patients, 81% complained of preoperative pain, and 66% of them reported total symptom relief after the surgical procedure. In one month, 54.6% of the patients presented a closure of epithelial defect, and half of the total group evolved with corneal thinning. The failure rate was 45.5% of the cases. CONCLUSION: Cryopreserved amniotic membrane transplantation can be considered a good alternative for treating refractory neurotrophic corneal ulcers, as it resulted in significant improvement in pain (66%) and complete epithelial closure (60%) in many patients at 1 month postoperatively. Notably, the high failure rate highlights the need for further studies to identify patientand ulcer-related factors that may influence the outcomes of this procedure.