[Research progress on imagine diagnosis of limbal stem cell deficiency].

Limbal stem cell deficiency (LSCD) is an ocular surface disease resulting from a reduction and/or dysfunction of limbal stem cells. The symptoms of LSCD are non-specific and can be difficult to distinguish from other ocular surface diseases through slit-lamp examination. Impression cytology is currently considered the gold standard for LSCD diagnosis; however,it is a qualitative method with low sensitivity. Nonetheless,emerging imaging techniques offer quantitative diagnosis and staging of LSCD. This review article examines four imaging methods and their associated parameters for diagnosing LSCD: optical coherence tomography,which measures corneal epithelial thickness; optical coherence tomography angiography,which detects corneal neovascularization; in vivo confocal microscopy,which measures corneal epithelial thickness,subbasal nerve density,and corneal basal cell density; and future applications of full-field/spectral-domain optical coherence tomography.

[Research progress on corneal organoids].

Corneal organoids are cornea-like tissue structures that are developed from induced pluripotent stem cells or embryonic stem cells in vitro. They have similar anatomical characteristics and gene expression profiles to the human cornea. Although the culture of corneal organoids is difficult, expensive, and time-consuming, they can be tailored to reconstruct the physiological environment and the interaction between corneal cells in each layer, which suggests broad application prospects. In this article, we review the latest research progress and summarize the application of corneal organoids in the research of corneal development mechanisms, keratopathy pathogenesis, donor corneal transplantation, and corneal organoids-on-a-chip, in order to provide reference for further clinical corneal research concerning keratopathy models, drug screening, and personalized medicine.

[Analysis of clinical manifestations and imaging characteristics of in vivo confocal microscopy for Nocardia keratitis].

Objective: To analyze the clinical manifestations and imaging characteristics of in vivo confocal microscopy (IVCM) for Nocardia keratitis. Methods: It was a retrospective case series study. Medical records of 16 consecutive patients (16 eyes) with Nocardia keratitis were collected from the Department of Ophthalmology at Beijing Tongren Hospital, Capital Medical University between 2018 and 2022. The group consisted of 11 males and 5 females. The inclusion criteria for the study were the presence of typical clinical manifestations of Nocardia keratitis and at least one positive pathogenic test (corneal scraping or microbial culture) indicating Nocardia infection. The medical history, clinical and microbiological examination data of the patients were analyzed, including risk factors, diagnosis time, clinical manifestations, diagnostic methods, strain isolation, cure time, and best corrected visual acuity before and after treatment. This study utilized techniques such as slit lamp microscopy, in vivo confocal microscopy (IVCM), scraping cytology, microbial culture, and mass spectrometry identification. Results: The main risk factors for Nocardia keratitis included plant or foreign body injuries (5 out of 16 cases), contact lens use (4 out of 16 cases), and surgery (2 out of 16 cases). The average time to diagnosis was (20.8±11.8) days, with the shortest time being 8 days and the longest being 60 days. The best corrected visual acuity was less than 0.05 in 7 patients, between 0.05 to 0.3 in 7 patients, and greater than or equal to 0.3 in 2 patients. The typical symptoms included superficial gray-white infiltration in a wreath-like pattern on the cornea, corneal ulcers with dry and gray-white necrotic tissue coverage, and in severe cases, corneal ulcer perforation. Nocardia corneal infection was identified in 12 out of 16 cases by scraping cytology, 9 out of 16 cases by mass spectrometry, and 8 out of 16 cases by both methods. IVCM showed the presence of fine and moderately reflective filamentous hyphae in the subepithelial and superficial stromal layer of the cornea, arranged in elongated, beaded, and branched structures. Infiltration of many hyper-reflective round inflammatory cells was also seen around the hyphae. Fourteen cases were treated with medication and 2 cases were treated with corneal transplantation. The average cure time was (37.5±25.2) days and there were no cases of recurrence during the follow-up period (all greater than 6 months). Conclusions: Nocardia keratitis is primarily characterized by dense, round, or wreath-like infiltration in the early stage, and by gray-white dry necrotic secretion and hypopyon on the surface of corneal ulcers in the middle and late stages. Fine, branched or beaded, and moderately reflective filamentous structures are the hallmark of the corneal lesion on the IVCM images.

[A case of bilateral corneal ulcer after left eye cataract extraction surgery].

The patient is a 73-year-old female who developed bilateral corneal ulcers one month after cataract surgery in her left eye. The diagnosis is bilateral conjunctival pemphigoid. She underwent a left-eye amniotic membrane transplant and a right-eye lamellar corneal transplant, and was treated with oral immunosuppressants. The patient's condition is stable.

[A case report of congenital osteogenesis imperfacta with corneal ectasia].

An 8-year-old male patient was admitted to ophthalmology for bilateral progressive blurred vision for 5 years. He had a history of multiple spontaneous fractures. Ocular examination revealed best-corrected visual acuity (BCVA) of 0.5 in the right eye and 0.6 in the left eye. Slitlamp examination showed bilateral blue sclerae, thining of the entire cornea and corneal ectasia. General physical examination demonstrated multi-site ligamentous laxity. The diagnosis of osteogenesis imperfacta was made. The patient was advised to wear rigid gas permeable contact lens with large diameter and stabilized peripheral curve, and the BCVA achieved 0.8 for both eyes.

[A case of bacterial keratitis after lamellar keratoplasty for pterygium].

A 48-year-old man presented to Beijing Tongren Hospital Ophthalmology Department with redness, increased secretions and vision loss in his right eye. He had been treated with pterygium excision and lamellar keratoplasty due to recurrent pterygium in the right eye. After corneal scraping and microbial culture, he was diagnosed as bacterial keratitis after pterygium lamellar keratoplasty in the right eye. After applying topical antibiotic eye drops, removing corneal graft and conducting amniotic membrane transplantation, corneal infection was controlled and his vision was recovered.

[Research progress of diagnosis in neuropathic corneal pain].

The cornea is the most innervated tissue in the human body. Neuropathic pain occurring in the cornea has gradually attracted the attention of ophthalmologists. However, the definition and pathogenesis of neuropathic corneal pain (NCP) have not been clearly defined, making the diagnosis and treatment of the disease extremely challenging. In recent years, with the application of ocular surface pain assessment scales, in vivo confocal microscopy and functional magnetic resonance imaging in the clinical assessment of NCP, the diagnostic methods of NCP have been enriched. This paper reviewed the research progress of diagnostic methods of NCP, with a view to improving the ophthalmologists' understanding of NCP and promoting the application of these technologies in the diagnosis of NCP.

[Detection of the spontaneous blinking pattern of dry eye patients using the machine learning method].

Objective: To establish a method to record the spontaneous blink pattern with a machine learning model, and to clarify the spontaneous blink pattern in patients with dry eye. Methods: It was a cross-setional study.We selected 357 dry eye patients (102 males and 255 females), aged (46.2±13.3) years, who visited corneal specialist clinics of Beijing Tongren Eye Center in 2019, as the dry eye group. The control group enrolled 152 normal controls, including 32 males and 120 females, aged (48.1±13.9) years. All participants completed the Ocular Surface Disease Index questionnaire, blink video capture, lipid layer thickness measurement, tear break-up time measurement, corneal fluorescein staining, and Schirmer Ⅱ test. Based on the assembled model built using UNet image segmentation algorithm and ResNet image classification algorithm, single frames of the blink video were analyzed, and then the palpebral opening height of each frame was obtained in order to establish a spontaneous blink wave. Finally, the characteristics of spontaneous blinks in dry eye patients were analyzed based on different types of complete blinks (types A, B and C) and partial blinks (types Ⅰ, Ⅱ and Ⅲ). Independent sample t test and Wilcoxon rank-sum test were used to judge if there was significant difference between the dry eye group and the normal group. Results: The accuracy of the segmentation model and the classification model was 96.3% and 96.0%, respectively, and the consistency with the manual analysis was 97.9%. In dry eye patients, the number of blinks was 30 (18, 42)/min, which was higher than that in normal controls [20 (9, 46)/min] (U=18 132.50, P=0.002). The number of complete blinks in dry eye cases was significantly lower than that in normal controls [6 (3, 24)/min vs. 12 (3,33)/min; U=12 361.00, P=0.016], and the number of partial blinks was significantly higher than that in normal controls [15 (6, 27)/min vs. 3 (0, 10)/min; U=22 839.00, P<0.001]. In complete blinks, the proportion of type A blinks in dry eye patients was significantly higher than that in normal controls [53.7% (2 796/5 177) vs. 39.3% (633/1 698); χ²=101.83, P<0.001]; in partial blinks, the proportion of type Ⅱ blinks in dry eye patients was significantly higher than that in normal controls [36.0%(2 334/6 477) vs. 29.6%(126/426); χ²=6.99, P=0.007]. The average interblink interval of dry eye patients was 1.2 s, which was not significantly different from that of normal controls (1.1 s; U=15 230.00, P=0.093). The eyelid closed phase of dry eye patients was 0.8 s, which was significantly shorter than that of normal controls (1.3 s; U=16 291.50, P=0.006). There were no significant differences in eyelid closing phase, early opening phase and late opening phase between the two groups (all P>0.05). Conclusions: In dry eye patients, the number of partial blinks increased, the number of complete blinks decreased, and the duration of eyelid closed phase shortened significantly. The main blink patterns of dry eye patients included type Ⅱ partial blinks with a reduced closure amplitude and type A complete blinks with a shortened closure time.

[Correlation of inflammatory cells and corneal nerve damage in fungal keratitis on in vivo confocal microscopy].

Objective: To investigate the relationship between inflammatory cell infiltration and nerve damage in patients with fungal keratitis at different degrees of severity. Methods: Retrospective study. A total of 44 consecutive patients (44 eyes) with fungal keratitis in Beijing Tongren Hospital Affiliated to Capital Medical University from January 2017 to December 2019 were selected as the patient group, including 30 males and 14 females, with an age of (58.3±11.5) years old. Twenty healthy people (20 eyes) were included as control group. Slit-lamp microscopy was performed to observe the corneal ulcer. According to the diameter of corneal ulcer, patients were divided into mild, moderate and severe groups. With in vivo confocal microscopic ,the images were obtained from the epithelial layer to the endothelial layer in the central cornea and superior, inferior, nasal and temporal peripheral cornea. Parameters of the maximum density of fungal hyphae, the maximum depth of hyphal infiltration, the density, area and length of dendritic cells (DCs), the nerve density, and the number and curvature of nerve trunks were collected. The Kruskal-Wallis test, Wilcoxon test, and Spearman correlation analysis were used for analyses. Results: On confocal microscopy, many uniform, highly reflective, segment-like structures in parallel or staggered rows were detected in the cornea, with a certain degree of physiological curvature and branching. Quantitative analysis of hyphal density found that the median rating of hyphal density was 2.6 (2.0, 3.0), mainly with medium to large amounts of hyphae. Most hyphae were 100-150 µm in depth (18 cases, 40.9%), and the maximum depth of hyphae in 95.5% (42 cases) of patients was within 300 µm. The hyphal invasion depth in the mild group was 89.4 (50.5, 106.8) µm, in the moderate group was 133 (122, 203) µm, and in the severe group was 135 (74, 151) µm. As the severity of the disease increased, the depth of hyphal invasion increased (F=4.248, P=0.001). Compared with the control group, the DC density [166 (81.3, 212.5) vs. 24.0 (20.8, 32.3) cells/µm2], area [441.3 (291.9, 529.5) vs. 63.7 (47.7, 70.3) µm2] and length [68.3 (39.4, 91.0) vs. 9.2 (7.0, 11.3) µm] increased in patients (W=493.5, 500.0, 500.0; P<0.01). The nerve density [5 398.3 (3 202.7, 6 828.3) vs. 19 171.8 (17 558.8, 21 550.4) µm/mm2; t=-14.448, P<0.01] and the length [692.7 (402.0, 925.1) vs.2 138.4 (1 940.4, 2 597.2) µm; t=-11.930, P<0.01] and number [2.9 (2.0,3.0) vs. 6.0 (5.5,7.0); t=-8.282, P<0.01] of nerve trunks in patients decreased. There were strong negative correlations between the nerve density, the number of nerve trunks, and the DC density (r=-0.555, -0.466; P<0.01). Conclusions: The depth of fungal hypha invasion in patients with fungal keratitis is mainly concentrated in the epithelial layer and superficial stroma layer. The density of mature dendritic cells in the lesion area was negatively correlated with the density and number of subbasal nerves. The density of subbasal nerves decreased as the increase of the severity of the lesion. (Chin J Ophthalmol, 2021, 57: 580-588).

[An interpretation of global consensus on the diagnosis and management of limbal stem cell deficiency].

Limbal stem cell deficiency is an ocular surface disease with the imbalance of corneal epithelial homeostasis caused by decrease of number or weakening of function of limbal stem cells. In 2019 and 2020, the international limbal stem cell deficiency working group developed and released the international consensus on the definition, classification, diagnosis, staging and management of limbal stem cell deficiency, which provided a standard protocol for related research and clinical diagnosis and treatment of limbal stem cell deficiency. In order to help Chinese ophthalmologists to properly understand its core components, there is a need to make in-depth interpretation to the key and difficult contents of the international consensus. (Chin J Ophthalmol, 2021, 57: 95-99).

[Research progress on limbal stem cell transplantation].

Limbal Stem Cell Deficiency (LSCD) is an ocular surface disease caused by the decrease of the quantity and dysfunction of limbal stem cell, which is characterized by conjunctivalization and other signs of epithelial dysfunction. For sever LSCD, surgery is the main treatment way. Recently, plenty of researches published the outcomes of different operation methods. This article summarized five major operations, including conjunctival limbal autograft (CLAU), simple limbal epithelial transplantation (SLET), limbal allograft, cultivated limbal stem cell transplantation (CLET) and cultivated oral mucosal epithelial transplantation (COMET). (Chin J Ophthalmol, 2020, 56:956-960).

[Progress of clinical diagnosis and treatment in fungal keratitis].

Fungal keratitis is an important cause of corneal blindness in China. Delayed diagnosis and treatment contribute to its poor prognosis. In recent years, with the advancement of laboratory test techniques, imaging diagnostic techniques and treatment methods, the diagnosis and treatment of fungal keratitis has constantly improved. This article reviews the recent progress of the laboratory and imaging diagnosis, medicine and surgical treatment in fungal keratitis. It may be helpful to promote the application of the new technologies in China and to improve the prognosis of fungal keratitis.(Chin J Ophthalmol, 2020, 56: 631-636).

[In vivo confocal microscopic characteristics of limbal stem cell deficiency].

Objective: To investigate the cellular changes and quantitative analysis of basal cell density (BCD) and corneal epithelial thickness (CET) in limbal stem cell deficiency (LSCD) by in vivo confocal microscopy (IVCM). Methods: Prospective case-control study. A total of 35 eyes of 23 patients diagnosed with LSCD and 25 eyes from normal subjects were included in this study. Based on slit-lamp presentation and the global consensus on classification, the LSCD patients were classified into LSCD Ⅰ, LSCD Ⅱ and LSCD Ⅲ. Confocal images of the central cornea, and the superior, inferior, nasal and temporal limbus were inspected by IVCM. Morphologic characteristics of LSCD were summarized. The BCD and CET in all locations were measured. ANVOA or Kruskal-Wallis test was used for analysis when appropriate. A receiver operating characteristic was used to detect the diagnosis efficiency of BCD and CET. Results: The characteristics in the corneal epithelium of LSCD on IVCM included nested corneal epithelial cells, goblet cells with hyper-reflective spots, irregular basal cells and decreasing subbasal nerve density. The mean BCD of the LSCD group was (8 976±1 096) cells/mm2 in the central cornea. Compared to the control group, the BCD in the central cornea, superior, inferior, nasal and temporal limbus decreased by 30.2%, 26.0%, 28.7%, 29.3% and 30.2%, respectively (all P<0.007). The CET in the central cornea was (47.3±8.1) µm. The CET in the central cornea, superior, inferior, nasal and temporal limbus decreased by 27.9%, 23.7%, 20.6%, 26.9% and 23.1%, respectively, compared to the control group (all P<0.007). There was a decline of BCD and CET in more serious LSCD. Additionally, the decline of BCD and CET was shown in the unaffected region. The receiver operating characteristic showed the diagnosis efficiency of BCD in the corneal center and limbus (0.931 and 0.916) was superior to CET (0.853 and 0.817). Conclusions: There was a series of characteristic cellular changes in LSCD on IVCM. Both BCD and CET decreased significantly in LSCD. The BCD had higher diagnostic efficiency for LSCD.(Chin J Ophthalmol, 2020, 56:447-455).

[Identification of pathogens in the vitreous of patients with infectious uveitis by metagenomic sequencing].

Objective: To investigate the role of metagenomic sequencing in the diagnosis of infectious uveitis. Methods: Cross-sectional study. A total of 19 vitreous specimens of patients with suspected infectious uveitis from March 2016 to July 2018 in Beijing Tongren Hospital were collected, including 8 males and 11 females, 19 to 68 years old. There were 10 cases in the right eye, 8 cases in the left eye and 1 case in both eyes. Acute retinal necrosis was clinically diagnosed in 8 patients (9 eyes), and the diagnosis was unknown in 11 patients (11 eyes). About 1 ml of the vitreous fluid was reserved for each specimen, 800 µl for metagenomic sequencing and 200 µl for real-time fluorescence quantitative PCR verification. The TIANamp Micro DNA Kit was used to extract the sample DNA for metagenomic sequencing, and the ultrasonic fragment was broken to 200-300 bp. The BGISEQ-500 platform was used for sequencing. The data with low quality and length less than 35 bp were cleared from the sequencing data to obtain high-quality data. Through biological authentication software, the reference human genome sequence and low complexity were removed from high-quality data. The data obtained were compared with a special microorganism database regarding the percentage of microbial sequences, the number of unique sequences, coverage and sequencing depth, so as to determine positive sequencing parameters, which were classified into bacteria, viruses, fungi and parasites. Real-time fluorescence quantitative PCR was performed to validate the accuracy. Results: A variety of microorganisms were detected by metagenomic sequencing in 19 specimens, including 3 cases of varicella zoster virus, 2 cases of Candida albicans, 1 case of Propionibacterium acnes and 1 case of Haemophilus parainfluenzae. The percentage of microbial sequences was 77.93% （1 794/2 302）, 99.98% （12 843/12 845） and 98.88%（5 733/5 798）, and the number of unique sequences was 1 794, 12 843 and 57 33 in varicella zoster virus cases, respectively. The verification of varicella zoster virus by PCR was consistent with that by metagenomic sequencing. Conclusion: Metagenomic sequencing can be used as an alternative method for laboratory diagnosis of infectious uveitis. (Chin J Ophthalmol, 2020, 56: 519-523).

Longitudinal changes in prostate volume in a community-based cohort study of middle-aged and elderly men in rural China.

BACKGROUND: Prostate volume (PV) and its change rate are important for the progression of prostate disease, but studies on their estimates are inconsistent. OBJECTIVES: To investigate whether age, prostate-specific antigen (PSA), and other specific characteristics are associated with PV and its change rate. MATERIALS AND METHODS: A community-based cohort study was conducted in a rural area of China among male residents aged 40-80 years. PV was estimated at baseline and at 4 years of follow-up by trans-abdominal ultrasound. Annual PV change rate (PVCR) was calculated as change in volume divided by time interval. Baseline characteristics, including age, serum PSA, and hormones, were evaluated. And their relationships with PV or PVCR were assessed with Pearson correlation and multivariate linear regression analyses. RESULTS: Totally, 462 participants completed the follow-up with baseline PV (PV0 ) of 15.6 ± 5.5 ml. PV0 was highly correlated with age and PSA in pairwise correlations (Pearson r = 0.35 and 0.34, respectively, p < 0.01). Multivariate linear regression showed similar associations that PV0 tended to increase with age and PSA. The average PVCR was 0.7 ± 1.8 ml/year. In pairwise correlations, PVCR was inversely correlated with PV0 and positively correlated with PSA, while it was not significantly related to baseline age. Linear regression of PVCR on age and PSA in groups classified by PV0 quartile showed that age was not a significant estimator of PVCR, whereas PSA was. In each PV0 group, PVCR tended to increase with PSA. DISCUSSION AND CONCLUSION: PV was positively associated with age and PSA, and it tended to grow faster in men with smaller baseline PV and higher PSA. PSA can be a valuable parameter for estimating both the size and the growth speed of prostate. Although age is associated with prostate enlargement, it does not appear to be related to the longitudinal change rate of PV.

[Diagnostic value of fungal fluorescence staining on corneal scrapings for fungal keratitis].

Objective: To analyze the sensitivity and specificity of fungal fluorescent staining in the diagnosis of fungal keratitis, and to compare it with conventional fungal culture, in vivo confocal microscopy (IVCM) and Giemsa staining. To explore its value of clinical application. Methods: Prospective case-control study. A total of 105 consecutive patients (105 eyes) diagnosed with infectious keratitis at Beijing Tongren Hospital from August 2017 to April 2018 were included. Patients with infectious keratitis were divided into fungal keratitis (FK) group and non-fungal keratitis (NFK) group by slit lamp microscopy, corneal in vivo confocal microscopy (IVCM) examination, and the results of Giemsa staining, fluorescent staining and pathogenic culture of corneal scraping from ulcer. The sensitivity and specificity of the above-mentioned examination methods for the diagnosis of fungal keratitis were analyzed. The receiver operating characteristic curve (ROC curve) and Area Under Curve (AUC) values were calculated to determine the diagnostic value of fungal fluorescent staining for fungal keratitis. Results: Among the 105 patients with infectious keratitis, 66 were fungal keratitis, 39 were non-fungal keratitis (29 cases of bacterial keratitis and 10 cases of acanthamoeba keratitis). Isolation from fungal keratitis were mainly Fusarium spp. (43.5%), followed by Alternaria spp. (21.7%) and Aspergillus spp. (19.6%). After fluorescent staining of the ulcer smear, the background of tissue demonstrated homogeneous black or weak blue fluorescence. The cell wall of fungi showed bright blue-violet to blue fluorescence, and the morphology, structure and hyphal density were easily recognized. The sensitivity of different methods for the diagnosis of corneal fungal infection were smear fluorescence staining (97.0%), IVCM (87.9%) , Giemsa staining (86.7%), and fungal culture (69.7%); the specificity of fungal culture was the highest (100%), followed by IVCM and Giemsa staining (94.9%), and fluorescent staining (87.2%). The ascending order of AUC values was: fungal culture (0.848)

[Therapeutic efficacy of ultraviolet combined with riboflavin for the rabbit bacterial keratitis].

Objective: To study the effects of ultraviolet light combined with riboflavin treatment (corneal collagen-crosslinking, CXL) on infectious control and stromal reconstruction of bacterial keratitis. Methods: Experimental Study. A Staphylococcus aureus rabbit keratitis model was established by injecting Staphylococcus aureus broth into the shallow stromal layer of the right eye cornea of New Zealand white rabbits. Forty-four rabbits that successfully established the model were randomly divided into four groups: corneal collagen cross-linking (CXL) group, antibiotic group, CXL+ antibiotic group and untreated group, with 11 rabbits in each group. Before the treatment and at 3, 7, 14 and 28 days after treatment, slit lamp corneal examination, AS-OCT and in vivo confocal microscopy (IVCM) were performed. Clinical efficacy of different treatments were evaluated at different time points. Parameters including conjunctival hyperemia, corneal ulcer, infiltration, edema, and neovascular. Histopathological examinations of corneal lesions were performed in order to detect the infiltration, inflammatory cells and repair in corneal tissue. Normal data were compared with paired t-test and non-normal data were compared with paired rank sum test before and after treatment. Kruskal-Wallis rank sum test was used to compare 4 groups of data and the generalized estimation equation is used to compare the repeated measurement data at each time point and the comparison between the groups of the treatment groups. Results: After treatment, different time points and specimens for pathological observation, we obtained the following results:Conjunctival hyperemia: in CXL and CXL+ antibiotic groups after treatment for 3 days from treatment before 3 (2, -4) and 3 (2, -3),The reduction was 2 (1, -3) and 2 (1, -2), the difference was statistically significant (Z=-3.91, -5.50; P<0.008); 14 days, the antibiotic group changed from 3 (3, -4) to 2 (1, -2) after treatment, the difference was statistically significant (Z=-5.11, P<0.008); the untreated group had no statistical significance before and after treatment. After 14 days of treatment, the area of corneal ulcer (0.08±0.11) cm(2), (0.07±0.05) cm(2) in CXL group and CXL+ antibiotic group was significantly lower than that before treatment (0.40±0.18) cm(2), (0.49±0.24) cm(2). The difference was statistically significant. Significance (Z=-3.29, -3.64; P<0.008); after 14 days of treatment, after 14 days of treatment, neovascularization in the CXL and CXL+ antibiotic groups began to resolve, 1 (1, -2) and 1 (0, -2) at 7 days of treatment. decreased to 1 (1, -1) and 0 (0, -1), the difference was statistically significant (Z=4.57, 3.80; P<0.012 5); The degree of corneal edema was significantly reduced in the CXL group and the CXL+ antibiotic group at 14 days after treatment, which was reduced from (650±154) µm and (785±255) µm before the treatment to (432±95) µm and (455±109) µm, the difference was statistically significant (t=4.50, 4.92; P=0.00); The density of corneal stromal cells was also reduced from (446±257)/mm(2), (321±145)/mm(2) to (107±66)/mm(2), (114±94)/mm(2), the difference was statistically significant (t=4.15, 4.76; P<0.05). Histopathological observation under light microscope showed that most of the corneal ulcers healed in the CXL group and the CXL+ antibiotic group at 7 days of treatment. The epithelial cells were clearly visible and misaligned, and a small amount of neutrophils in the stromal layer. The upper epithelial layer was treated for 14 days. The cells are arranged neatly, the structure is clear, and the inflammatory cells are significantly reduced. Conclusion: Ultraviolet light combined with riboflavin corneal collagen cross-linking has a certain therapeutic effect on rabbit bacterial keratitis infection control and ulcer repair, and can be used as an auxiliary treatment for antibiotics. (Chin J Ophthalmol, 2018, 54:902-910).

[Genetic typing and susceptibility testing of strains from Staphylococcus aureus keratitis or conjunctivitis patients].

Objective: To study the relationship between genetic typing and the antibiotic susceptibility of staphylococcus aureus (SA) isolated from keratitis or conjunctivitis patients. Methods: Experimental study. Thirty-four (34) strains of Staphylococcus aureus were isolated from 34 cases of keratitis or conjunctivitis. The genomic DNA was extracted and amplified with PCR. With the method of multi locus sequences typing (MLST), gene fragments from 7 house-keeping genes were amplified and the products were sequenced. The results were submitted to the MLST website (www.pubmlst.org). In comparison with the allele of the corresponding gene, the allele spectrums of the strain were obtained with 7 housekeeping genes. At last, the MLST genotypes of the isolated strains were determined. With the START software, the evolutionary tree was established with UPGMA method. With the microdilution method, the MIC(90) of 13 antimicrobial agents was determined. The MIC(90) value of antimicrobial agents among different genotypes of Staphylococcus aureus was comparatively analyzed. Results: Ten (10) genotypes were obtained from 34 strains of Staphylococcus aureus. The dominant types were ST239, ST2592 and ST188. The clustering of genotyping was relatively concentrated, mainly in group Ⅰ (25 strains of SA, 83.3% of the total), and followed by group Ⅱ (5 strains of SA, 16.7%). The conjunctival isolates were distributed in the subgroup A of group Ⅰ. The cornea isolates were concentrated in subgroup B and group Ⅱ. With the exact probability method, the R×C chi square tests were used as statistic analysis method. The difference between the bacterial genotyping of two sources was statistically significant (P=0.011). Twenty-four strains of SA in group Ⅰ was sensitive to Vancomycin, Rifampicin and Amikacin (sensitivity ratio was 24/24, 20/24 and 20/24, respectively), and was generally resistant to other antibiotics. The values of MIC(90) of ciprofloxacin, ofloxacin, gatifloxacin and moxifloxacin to Staphylococcus aureus in subgroup A (0.16±0.07, 0.51±0.42, 0.31±0.14, 0.22±0.33) were significantly lower than the values in subgroup B(0.74±0.11, 0.84±0.45, 0.67±0.03, 0.68±0.26). The difference was statistically significant (P=0.004, 0.026, 0.034, 0.001). There was no significant difference between the MIC(90) values of the other 9 kinds of antibiotics in the subgroup A and in the subgroup B of Staphylococcus aureus (P value 0.047-0.561). Conclusion: The genotype of Staphylococcus aureus of corneal isolations and conjunctival isolations were different. The conjunctival isolates were distributed in the subgroup A of group Ⅰ and the corneal isolates were concentrated in subgroup B and group Ⅱ. There is a significant correlation between the MLST genotypes and antibiotic sensitivity. (Chin J Ophthalmol, 2018, 54:767-774).

[Histopathology manifestation and imaging characteristics of in vivo confocal microscopy for diagnosis of ocular surface squamous neoplasia].

Objective: To study the histopathology manifestation and imaging characteristics of in vivo confocal microscopy (IVCM) for diagnosis of ocular surface squamous neoplasia (OSSN) and analyze the reliability of IVCM in differential diagnosis from OSSN cases. Methods: A prospective study. Twenty-three patients (23 eyes) with OSSN were collected from September 2015 to November 2017 in Beijing Tongren Eye center. They were underwent the examinations sequentially as follows: visual examination; slit-lamp microscope examination and ocular surface photography; color fundus image; anterior segment optical coherence tomography examination; IVCM examination and histopathology examination after surgery. With histopathology diagnosis, all subjects were divided into two groups: conjunctival intraepithelial neoplasm (CIN) and squamous cell carcinoma (SCC). Compared with histopathological findings, the IVCM results (cell morphology, cytoplasm and nucleus) of OSSN were analyzed. The difference analysis between the count data of two groups was carried out by the chi square test or Fisher's exact test. Receiver operating characteristic curve and area under the curve (AUC) were used to describe the accuracy of each parameter to differentiate SCC from OSSN. Results: There were 12 males and 11 females among the 23 patients aged (62±15) years. With the histopathological diagnosis, there were 15 cases (15 eyes) in the CIN group and 8 cases (8 eyes) in the SCC group. Eye redness, pain, foreign body sensation and tears were the symptoms of OSSN. There was no significant difference in clinical symptoms between the CIN and SCC groups. The size of tumors and the incidence of trophoblastic vessels in the SCC group were greater than those in the CIN group [8.5(6.5-15.5) mm vs. 5.8(4.0-8.5)mm, Z=4.702,P=0.029; 7/8 vs. 5/15, χ2=6.135, P=0.013). In histopathology, multilayered epithelia with cellular polymorphism, varied cytoplasm staining, and slightly thicker nuclei were observed in the CIN group. The corneal epithelial cells of SCC patients were markedly heterogeneous, and the intercellular bridge structure can be detected. Proliferative cells appeared with spindle shaped cells, nuclear mitotic figures and syncytial cells in the SCC group. The IVCM examination in the CIN group and SCC group showed that the cytoplasm was high and the ratio of nuclear plasma increased. There was no significant difference between the two groups in the size of epithelial cells, cytoplasm reflectivity, and nuclear cytoplasm ratio (P=0.053, 0.108, 0.067). The proportions of nuclear mitotic figures, spindle or chimeric cells, nests of whirlpool cells and the abnormal cells of the superficial stroma layer (8/8, 8/8, 8/8 and 8/8) in the SCC group was higher than those (2/15, 1/15, 4/15 and 0/15) in the CIN group, and the difference was statistically significant (P=0.001, 0.001, 0.003; P<0.001). Among these IVCM parameters, the abnormal cells of the superficial stroma layer had the highest diagnostic ability with the AUC value of 1.000, and the following AUC values were 0.933 for mitotic or double nuclei, 0.901 for spindle or chimeric cells, and 0.867 for the nests of whirlpool cells. Conclusions: The IVCM characteristics of OSSN are the dysplastic cells, hyper-reflective cytoplasm and increased nuclear plasma ratio in the corneal epithelium. Dysplastic cells appearing in the superficial stroma layer, nuclear mitotic figures and nests of vortex cells are the main IVCM parameters to support the malignant change of OSSN lesions. (Chin J Ophthalmol, 2018, 54: 652-660).

[Application of clustered regularly interspaced short palindromic repeats- associated protein 9 gene editing technology for treatment of HBV infection].

A lack of effective drugs and technical means to eradicate hepatitis B virus (HBV) is a bottleneck that limits the ability to fully cure HBV infection. Recently, genome-editing technology based on clustered regularly interspaced short palindromic repeats -associated protein 9 is an emerging technique for editing specific gene loci, which can specifically target HBV covalently closed circular DNA, effectively inhibits HBV DNA replication and regulates HBV functional protein expression, and is expected to become a powerful gene therapy tool for the complete eradication of HBV. Considering this, it has become the focus of attention for scholars at home and abroad that how to use clustered regularly interspaced short palindromic repeats -associated protein 9 to accomplish modification of HBV genomes for complete eradication of HBV. This paper summarizes the latest progress based on the latest research results at home and abroad in the application of clustered regularly interspaced short palindromic repeats -associated protein 9 gene editing technology in anti-HBV infection treatment, and expounds its potential and challenges as a radical cure for HBV infection.