Application of Metagenomic Next-Generation Sequencing in the Diagnosis of Infectious Keratitis.

Purpose: To determine the advantages of next-generation metagenomic sequencing (mNGS) technology in the diagnosis and treatment of infectious keratitis (IK). Methods: A total of 287 patients with IK admitted to the Department of Ophthalmology of Nanjing First Hospital between August 2018 and December 2022 were analyzed retrospectively, and the pathogenic causes, etiological characteristics, detection, treatment methods, and efficacy were summarized. Results: Trauma and foreign matter were the most common causes of IK (144 patients, 50.2%). Of the 287 patients, 228 (79.4%) were diagnosed with a specific etiology, including 110 (48.2%) fungal infections, 44 (19.3%) viral infections, 42 (18.4%) mixed infections, and 30 (13.2%) bacterial infections. Filamentous fungi represented by Fusarium and Aspergillus were the most common, followed by bacteria such as Pseudomonas aeruginosa, Streptococcus pneumoniae, viruses (Herpes Simplex Virus/Varicella-Zoster Virus), and parasites. The positivity rates of secretion culture, corneal laser confocal microscopy (CM), mNGS, and pathological sections were 47.3% (133/281), 45.3% (111/245), 83.9% (104/124), and 19.3% (40/207), respectively. The positivity rate of mNGS for bacteria and viruses was higher than that of the other methods, and the positivity rate for fungi was the same as that for CM. As a result, 214 cases (74.6%) were cured, 51 cases (17.8%) improved, 8 cases (2.8%) did not heal, ocular content enucleation was performed in 14 cases (4.9%), and the overall efficacy rate was 92.3%. Conclusion: Trauma and foreign matter are the main causes of IK. The mNGS technology is an efficient and comprehensive detection method for viruses and bacteria, especially for mixed infections.

Advances in the application of corneal confocal microscopy / 国际眼科杂志(Guoji Yanke Zazhi)

·Corneal confocal microscopy can be used in the real-time, noninvasive, high-resolution corneal structure at the cellular level, which has been widely used in corneal disease research. This review summarizes recent advances in corneal confocal microscopy in the study of infectious keratitis, dry eye, keratoconus, diabetic peripheral neuropathy, and clinical studies to guide corneal transplantation.

Real-time polymerase chain reaction for the diagnosis of necrotizing herpes stromal keratitis.

AIM: To design, optimize and validate a rapid, internally controlled real-time polymerase chain reaction (RT-PCR) test for herpes simplex virus (HSV) in the diagnosis of necrotizing herpes stromal keratitis. METHODS: Tears alone or together with corneal epithelium scrapings from 30 patients (30 eyes) suspected of necrotizing herpes stromal keratitis were tested for HSV DNA by RT-PCR. The samples were collected during the first visit and then on the subsequent 7, 14, 28, 42, and 56d. The symptoms of the patients were scored before treatment to determine the correlation between HSV concentration in the corneal epithelium scrapings and clinical scores. RESULTS: The positive rate (46.4%) in the corneal epithelium group before the therapy was significantly higher than that (13.3%) in the tears group (P=0.006). There were 13 positive HSV patients before the therapy, the concentration of HSV DNA in corneal epithelium scrapings group was significantly higher than that in the tears group (paired t-test, P=0.0397). Multilevel mixed-effects model analysis showed that the difference between the corneal epithelium scrapings group and the tears group was statistically significant (P=0.0049). The Spearman rank correlation analysis indicated a positive correlation between the HSV concentration in the corneal epithelium scrapings and clinical scores before the treatment (r=0.844, P<0.0001). CONCLUSION: RT-PCR appears to be a powerful molecular tool for the diagnosis of necrotizing herpes stromal keratitis.

Identification of corneal neovascularization-related long noncoding RNAs through microarray analysis.

PURPOSE: To reveal the role of long noncoding RNAs (lncRNAs) in corneal neovascularization (CN). METHODS: We established a murine CN model and performed lncRNA expression profiling to identify differentially expressed lncRNAs between normal and vascularized corneas. Based on Pearson correlation analysis, an lncRNA/mRNA coexpression network was constructed. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of lncRNA-coexpressed mRNAs were conducted to determine the related biological modules and pathological pathways. Real-time polymerase chain reactions were carried out to detect the expression pattern of lncRNA in the clinical samples. RESULTS: A total of 154 differentially expressed lncRNAs were identified between vascularized and normal corneas, including 60 downregulated lncRNAs and 94 upregulated lncRNAs. GO enrichment analysis of lncRNA-coexpressed mRNAs indicated that the biological modules were correlated with extracellular region, DNA binding, and immune response. KEGG pathway analysis indicated that "pathways in cancer" was the most enriched signaling pathway. Moreover, the human ortholog of NR_033585 and lincRNA:chr8:129102060-129109035 reverse strand was found to be differentially expressed between vascularized and avascular corneas. CONCLUSIONS: This study provides a novel insight into CN pathogenesis. The intervention of dysregulated lncRNAs may become potential targets for the prevention and treatment of ocular vascular diseases.

Liquid nitrogen-cryopreserved tarsal plate and palpebral conjunctival transplantation for the repair of tarsal defects.

The objectives of this study were to compare the efficiency of three methods for repairing tarsal defects (glycerine-preserved, alcohol-preserved and cryopreserved tarsal plate and palpebral conjunctival transplantation) based on histopathological changes and apoptosis, and to evaluate the clinical effects of repairing tarsal defects by liquid nitrogen-cryopreserved tarsal plate and palpebral conjunctival transplantation. Forty-eight rabbit eyes with tarsal defects were equally divided into three groups and transplanted with tarsal plates and palpebral conjunctivae under the following conditions: Group 1, liquid nitrogen cryopreservation; Group 2, glycerine preservation; and Group 3, alcohol preservation. Histopathological changes in the transplants were observed by light microscopy 1 week, 1 month and 3 months postoperatively. Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Clinically, transplantations of liquid nitrogen-cryopreserved tarsal plates and palpebral conjunctivae were performed in 30 eyes (29 cases) to repair tarsal defects caused by the excision of neoplasms. The postoperative inflammatory reaction and number of apoptotic cells in Group 1 were lower compared with those in Groups 2 and 3. Clinically, of the 30 eyes operated on, 14 were cured, 15 improved and 1 failed between 6 and 90 months of follow-up. Liquid nitrogen-cryopreserved tarsal plate and palpebral conjunctival transplantation is an easy, feasible and convenient procedure. Its effects are fairly favorable, with only a small rejection rate postoperatively. Therefore, it is an ideal method for repairing tarsal defects.

Awareness of cornea donation of registered tissue donors in Nanjing.

OBJECTIVE: To evaluate the current cornea donation awareness of tissue donors in the city of Nanjing, China. METHODS: Altogether 2000 registered tissue donors in the Red Cross Eye Bank of Nanjing by the end of 2010 and 2000 control residents of Nanjing in February to June 2011 were randomly selected to participate in our field questionnaire survey. The questionnaire consisted of questions regarding the understanding of cornea donation, the attitude toward cornea donation, and attitude toward legislation and free donation. The awareness of cornea donation between the registered tissue donors and residents was compared. Related factors of the willing-ness to donate corneas and to become a tissue donor were evaluated with univariate and multiple logistic regression analysis. RESULTS: A total of 1867 (response rate: 93.4%) tissue donors and 1796 (response rate: 89.8%; effective questionnaires: 1697) residents participated in this survey. For the questions about the knowledge of cornea donation, 90.3% tissue donors (residents: 78.9%) knew that donated corneas could be used for transplantations; 71.2% tissue donors (residents: 47.6%) knew that the appearance would not be destroyed after cornea donation; 70.7% tissue donors (residents: 20.0%) knew the formalities to become a cornea donor. For attitude toward cornea donation, 82.2% tissue donors (residents: 45.1%) were willing to donate corneas or eyeballs after death; 84.0% tissue donors (residents: 30.2%) had discussed with their families about donation; 85.1% tissue donors (residents: 24.8%) supported their families' or friends' cornea donation. For attitude toward legislation and free donation, 88.3% tissue donors (residents: 61.3%) approved of legislation to regular cornea donation; 72.2% tissue donors (residents: 38.8%) thought that cornea or organ donation should be gratis. The difference between two groups was significant (P<0.001). However, some tissue donors did not know cornea donation well, some even opposed the legislation of cornea donation and free donation protocol. For the factors influencing the willingness to donate corneas, population was the most relevant factor (P<0.001), since tissue donors were more willing to donate corneas than residents. Other significantly related factor were age, gender, occupation, and education level (P<0.001), while political status or religion were not significant. For the factors related to becoming a tissue donor, older age, male, white-collars (including white-collar, civil servant, teacher, soldier, lawyer, salesman, healthcare provider), higher education level, party members (including the Chinese Communist Party members, democratic party members, and the Communist Youth League members) (P<0.05). CONCLUSIONS: The registered tissue donors have better understanding and more positive attitude towards cornea donation than control residents do. They approve of the legislation of cornea donation and free donation protocol. It may be more effective to target the population of over 58 years old, male, white-collars, well-educated people, and party members in the promotion of cornea donation.

Mitomycin C "straight scleral tunnel incision"--trabeculectomy with a releasable suture.

OBJECTIVE: To evaluate the efficacy of "straight scleral tunnel incision" -trabeculectomy with a releasable suture supplemented with mitomycin C (MMC) on reducing intraocular pressure (IOP), complications, and corneal astigmatism in patients with primary angle-closure glaucoma (PACG). METHODS: Totally 217 acute or chronic PACG patients with occludable angle above 180 degrees and IOP above 21 mm Hg were divided into 3 groups. Patients in group A (98 cases, 128 eyes), B (71 cases, 95 eyes), and C (48 cases, 60 eyes) were treated with "straight scleral tunnel incision"-trabeculectomy with a releasable suture supplemented with MMC, releasable suture trabeculectomy with MMC, and trabeculectomy with MMC, respectively. IOP, complications, and surgically induced astigmatism (SIA) were evaluated preoperatively and up to 12 months postoperatively. RESULTS: IOP of 2 weeks after treatment was significantly lower than preoperative IOP in all the 3 groups (all P <0. 001). Success rates (IOP < or = 20 mm Hg) in group A, B, and C were 87.91%, 89.23%, and 83.72% respectively at 12 months after treatment (P = 0.256). The incidence of shallow anterior chamber and hypotony had no significant difference between group A and B, but both of them were lower than that in group C (P < 0.05). There were no significant differences in preoperative corneal astigmatism among the 3 groups. The corneal astigmatism after 2 weeks in group A (1.71 +/- 1.47D) was higher than that before operation (1.28 +/- 1.05D, P = 0.126). With 12 months gone, the astigmatism almost returned to preoperative levels. The corneal astigmatisms after 2 weeks in group B and C (1.99 +/- 1.20D and 2. 22 +/- 1.39D) were significantly higher than those before operation (1.20 +/- 0.85D and 1.18 +/- 0.93D, P = 0.002, P = 0.001), respectively. With 12 months gone, the mean astigmatisms in group B and C (1.87 +/- 0.91D and 1.90 +/- 1.16D) were still significantly higher than those before operation (P = 0.001, P = 0.003). The highest astigmatic polar values in group A, B, and C (1.00D, 1. 89D, and 1. 77D) occurred after 2 weeks, 1 month, and 1 month postoperation, respectively, which were significantly higher than those before operation (0.19 +/- 1.32D, 0.12 +/- 1.22D, and 0.17 +/- 1.25D, P < 0.01), respectively. With 12 months gone, they were 0. 03D, -0. 18D, and -0. 13D higher than those before operation, respectively. The rates of function bleb and thin-wall bleb were 71.43% and 26.37% in group A, 75.38% and 29.23% in group B, 72.09% and 25.58% in group C, respectively at 12 months after treatment There were no significant differences among the 3 groups. CONCLUSION: "Straight scleral tunnel incision" -trabeculectomy with a releasable suture supplemented with MMC can reduce complications and get satisfactory results in reducing IOP and SIA.