Interactions between the constitutive host defences of tears and Staphylococcus epidermidis

As part of the normal microbiota of the external eye, Staphylococcus epidermidis has probably developed strategies to overcome tear defences. The interaction of this bacteria with constitutive tear proteins was examined. Isolates of S. epidermidis grown in 10% serum or artificial tear fluid containing lysozyme or lactoferrin were resistant to the action of these proteins. Using ELISA, cell wall binding of C3, vitronectin and lactoferrin differed quantitatively between strains and in closed-eye compared to open-eye conditions. No differences were observed between ocular and non-ocular strains. This suggests that ocular isolates originate from the general host microbiota and S. epidermidis isolates are resistant to individual constitutive tear proteins.     

Effect of lysozyme on adhesion and toxin release by Staphylococcus aureus.

PURPOSE: Staphylococcus aureus is a common cause of ocular infection and inflammation. We hypothesized that potential for S. aureus to cause an ocular infection would be enhanced if these bacteria are able to adhere to the biomaterials used in contact lenses. In turn, bacterial adhesion could also be influenced by other factors, such as properties of contact lenses and the absorbance of some tear components. We investigated the effect of the tear protein lysozyme on S. aureus adhesion to contact lenses and its effect on production of toxins or enzymes. METHODS: Bacterial adhesion on contact lenses was determined by counting the total number of bacteria as well as viable bacteria on lysozyme-coated or non-coated lenses, and by counting bacteria grown in the presence or absence of lysozyme in the medium. Toxin and enzyme production was assessed by haemolysis and proteolysis assays. RESULTS: Our results indicate that adhesion was significantly increased in the presence of lysozyme, both in the medium and coated onto contact lenses (P=0.04). The presence of lysozyme did not alter the production of alpha- or beta- toxins. However, the presence of lysozyme inhibited elastase activity. CONCLUSION: These results indicate that lysozyme deposition on contact lenses promoted S. aureus adhesion. The tear protein lysozyme might modify elastase activity and thus modulate the production of corneal degradation resulting from the action of this enzyme.     

Bacterial invasion of corneal epithelial cells.

PURPOSE: The normal ocular surface is frequently colonized by commensal gram-positive species. Gram-negative bacteria are often implicated in corneal infection and inflammation, particularly in association with soft contact lens wear. The aim of this study was to elucidate possible mechanisms of virulence in ocular bacteria. METHODS: The susceptibility of a human corneal epithelial cell line to bacterial invasion and association was evaluated using the gentamicin exclusion assay. Organisms tested included isolates from corneal ulcers, corneal inflammation and ocular sites in asymptomatic individuals. RESULTS: The commensal, non-pathogenic bacterium Staphylococcus epidermidis and some pathogenic strains of Serratia marcescens did not invade corneal epithelial cells. In contrast, pathogenic strains of Pseudomonas aeruginosa associated with and invaded corneal epithelial cells. CONCLUSIONS: The increased association of P. aeruginosa, compared to other bacterial types, might be a reason for the more frequent association of this bacterium with contact-lens-associated microbial keratitis.     

A practical treatment algorithm for managing ocular surface and tear disorders

Management of ocular surface irritation and morbidity associated with dry eye has been plagued by the complex interplay of different pathogenic elements and substantial variability of ocular surface deficits in patients. A practical algorithm is proposed to achieve effective management of dry eye. When the eye is open, ocular surface health is governed by a stable tear film that is maintained by neuroanatomic integration via 2 reflexes. Any dysfunctional element in this neuroanatomic integration is potentially pathogenic and creates ocular surface deficits leading to dry eye. In general practice, 5 major dysfunctional elements have been identified: decreased ocular surface sensitivity, aqueous tear deficiency, lipid tear deficiency, delayed tear clearance, and ineffective tear spread. Clinical workup should be individualized to identify all such dysfunctional elements in each patient through history taking, external and slit-lamp examination, and special tests. However, practical management lies in the detection of delayed tear clearance. The following strategies are advised: (1) eliminate all intrinsic inflammatory, infectious, allergic, and toxic insults, especially those associated with delayed tear clearance; (2) correct diseases that impede and interfere with tear spread and capacity; (3) create delayed tear clearance for aqueous tear-deficient dry eye by punctual occlusion; and (4) treat lipid-deficient dry eye after sufficient aqueous tears have been conserved. The aforementioned algorithm ameliorates ocular surface irritation and curtails morbidity in most patients. This algorithm can also be adopted for complex cicatricial ocular surface diseases before managing the remaining deficits resulting from hydrodynamic deficiency.     

Lacritin and other new proteins of the lacrimal functional unit

The lacrimal functional unit (LFU) is defined by the 2007 International Dry Eye WorkShop as ‘an integrated system comprising the lacrimal glands, ocular surface (cornea, conjunctiva and meibomian glands) and lids, and the sensory and motor nerves that connect them’. The LFU maintains a healthy ocular surface primarily through a properly functioning tear film that provides protection, lubrication, and an environment for corneal epithelial cell renewal. LFU cells express thousands of proteins. Over 200 new LFU proteins have been discovered in the last decade. Lacritin is a new LFU-specific growth factor in human tears that flows through ducts to target corneal epithelial cells on the ocular surface. When applied topically in rabbits, lacritin appears to increase the volume of basal tear secretion. Lacritin is one of only a handful of tear proteins preliminarily reported to be downregulated in blepharitis and in two dry eye syndromes. Computational analysis predicts an ordered C-terminal domain that binds the corneal epithelial cell surface proteoglycan syndecan-1 (SDC1) and is required for lacritin's low nanomolar mitogenic activity. The lacritin-binding site on the N-terminus of SDC1 is exposed by heparanase. Heparanase is constitutively expressed by the corneal epithelium and appears to be a normal constituent of tears. Binding triggers rapid signaling to downstream NFAT and mTOR. A wealth of other new proteins, originally designated as hypothetical when first identified by genomic sequencing, are expressed by the human LFU including: ALS2CL, ARHGEF19, KIAA1109, PLXNA1, POLG, WIPI1 and ZMIZ2. Their demonstrated or implied roles in human genetic disease or basic cellular functions are fuel for new investigation. Addressing topical areas in ocular surface physiology with new LFU proteins may reveal interesting new biological mechanisms and help get to the heart of ocular surface dysfunction.     

新型OxyPlateTM厌氧系统隔离眼部厌氧菌的评估

目的:厌氧细菌可引起眼部感染,我们测试OxyPlate^TM厌氧系统(OxyPlate^TM Anaerobic System,OXY)隔离可引起眼部疾病的厌氧细菌。方法:OXY不需要直接的厌氧条件（比如厌氧袋,罐）,将其与常规的厌氧袋培养基相比。琼脂培养基上眼部厌氧细菌菌株在好氧和厌氧条件下（厌氧袋）行标准的菌落计数:（1）OXY（好氧）;（2）5%羊血（sheep blood,SB）;（3）巧克力琼脂;（4）Schaedler琼脂。测试的眼部体外培养细菌来自眼内炎,泪囊炎,包括10个丙酸杆菌和3个放线菌种类。在每个培养条件下,每个细菌菌落计数隔离,排名从大到小,并在非参数比较下确定最佳的培养条件。结果:所有的厌氧条件对于厌氧菌株呈阳性反应。厌氧菌在有氧条件下的SB和Schaedler的琼脂中无法增长。痤疮丙酸杆菌在巧克力琼脂中生长稀疏。作为一种厌氧系统,在厌氧袋SB分离比OXY（P=0.0028）和巧克力琼脂（P=0.0028）分离出更多的菌落数。结论:虽然OXY经测试并没比其他的厌氧系统更高效,它似乎是一个合理隔离厌氧细菌的替代方法。其琼脂培养基在一个专门设计的盘并不需要厌氧袋使得OXY优于其他厌氧系统。     

Tear proteins of normal young Hong Kong Chinese

Background: Analysis of tear proteins is of diagnostic value for abnormal ocular conditions such as dry eye syndrome. Many studies of tear proteins have been performed on Caucasian subjects. However, little is known about these proteins in Chinese eyes. Methods: The total tear protein concentrations of 30 normal young Hong Kong Chinese were determined by the Bradford method and the modified Lowry method. Bovine serum albumin (BSA) and bovine immunoglobulin G (IgG) were both used as standards for each method. The tear protein patterns were determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and the concentrations of major tear proteins were quantified by scanning densitometry after SDS-PAGE. Results: The mean±SD total tear protein concentrations determined by the Bradford method, using BSA and IgG as standards, were 6.05±1.58 mg/ml and 11.48± 2.32 mg/ml respectively. The values determined by the modified Lowry method, using the same two standards, were 9.66±2.03 mg/ml and 7.53±1.80 mg/ml respectively. The mean±SD concentrations of major tear proteins were 2.73±0.82 mg/ml for lactoferrin, 0.021±0.028 mg/ml for human serum albumin, 2.89± 0.88 mg/ml for tear-specific prealbumin and 2.46±0.44 mg/ml for lysozyme. Conclusion: The results of total tear protein concentrations indicated that values obtained from different methods and different standards were not comparable. The tear protein patterns of our subjects were qualitatively similar to those reported for Caucasian subjects. However, the concentrations of the major proteins of our subjects were not in accordance with those reported previously. The main reason may be the large variability of method used. Received: 13 September 1999 Revised: 19 January 2000 Accepted: 25 January 2000     

Frequent association of delayed tear clearance in ocular irritation

AIM—To explore the pathogenic role of delayed tear clearance.
METHODS—By comparing 10 patients with punctal obstruction and 20 asymptomatic normals, delayed tear clearance was diagnosed in 70 patients without apparent punctal obstruction using fluorescein clearance test.
RESULTS—The majority were older (71.4 (SD 1.2) years) and women (66%). Frequent complaints included redness, itching, mucus discharge, and crusting, which tended to be worse upon awakening. Common associated problems were medicamentosa (13%), drug induced pseudo pemphigoid, ocular hypertension (27%), and glaucoma (7%). Topical non-preserved 1% methylprednisolone resulted in subjective (83%) and objective (80%) improvement and resolution of delayed tear clearance (87%).
CONCLUSION—These results indicate strong association of delayed tear clearance with intrinsically and extrinsically generated ocular surface inflammation. The presence of delayed tear clearance may set up a vicious cycle to aggravate the existing inflammation. Future prospective studies are needed to delineate the pathogenic role of delayed tear clearance in various ocular surface disorders.

Keywords: medicamentosa; ocular irritation; tear clearance; tear turnover     

Tear concentrations of azithromycin following topical administration of a single dose of azithromycin 0.5%, 1.0%, and 1.5% eyedrops (T1225) in healthy volunteers

PURPOSE: To evaluate azithromycin tear concentrations after one drop of T1225 0.5%, 1.0%, and 1.5% eyedrops. METHODS: In this randomized, double-masked study, 91 healthy volunteers received one drop into each eye of T1225 0.5% (n=23), T1225 1.0% (n=38), or T1225 1.5% (n=38). Azithromycin tear concentrations were measured by HPLC-MS at seven time points for 24 hours. Tolerability was evaluated. RESULTS: T1225 1.0% and 1.5% had similar pharmacokinetic profiles. After a post-instillation peak (167 to 178 mg/L after 10 minutes), mean concentrations remained above 7 mg/L for 24 hours (except for T1225 1% at H24). A delayed increase of the azithromycin mean tear concentration might be explained by the known late azithromycin release from tissues after storage in cells. Areas under inhibitory curve (AUICs) of T1225 1.0% and 1.5% were higher than AUICs of T1225 0.5% and ranged between 47 and 90. The three T1225 concentrations were safe for the ocular surface. CONCLUSIONS: Once daily instillation of T1225 1.0% and 1.5% was shown to reach an AUIC markedly above the required threshold for an antibacterial activity against Gram-positive bacteria (25-35). These results suggest that a BID instillation is more likely to ensure antimicrobial activity against Gram-negative bacteria (threshold >100).     

Role of tear fluid in the growth of Gram-negative bacteria on contact lenses

Incidents of CLARE (contact lens induced acute red eye) are associated with high numbers of Gram-negative bacteria on the lens surface. This study found that most strains of bacteria implicated in CLARE adhere poorly to the lens surface regardless of whether the lens surface was clean or had been worn in the eye. The high numbers of bacteria on the lens surface result from bacterial growth on the lens surface after adhesion has occurred. Using an artificial tear formulation and real tears, this study demonstrated that the tear fluid provides the necessary nutrients required for bacterial growth.     

多重细菌感染性眼病病原学分析

目的回顾性分析眼科细菌培养中多重病原菌的分布及变化。设计回顾性研究。研究对象眼部细菌培养标本。方法对北京同仁医院眼科1994～2004年眼部7786份标本进行细菌分离培养，对多重感染的标本培养结果进行回顾性分析。主要指标多重细菌培养阳性率、病原菌眼部分布情况及其耐药性。结果细菌培养阳性者1966株，其中多重感染者393株（患者191例）（20．0％）。在多重感染者中，革蓝阳性与阴性球菌分别占51．7％、6．1％；革蓝阳性与阴性杆菌分别占23．4％、18．8％。在多重感染者中，表皮葡萄球菌占首位（23．6％），其次为棒状杆菌属（21．8％）。眼部多重感染者多数为2种细菌的感染（94．8％），少数为3种或3种以上细菌感染。多重细菌感染的标本分布为结膜占54．5％，角膜占19、4％，玻璃体及房水占6．3％，泪器及其他部位占19．8％。儿童占27．8％，成年人占52．9％，老年人占19．3％。393株培养阳性细菌对氧氟沙星、环丙沙星、庆大霉素及妥布霉素的敏感率分别为78．6％、78．2％、72．0％及62．2％。结论多重细菌感染者以两种细菌同时感染为主，多数菌株对氧氟沙星和环丙沙星敏感。（眼科，2007，16：55-58）     

Variability of tear protein levels in normal young adults: between-day variation

Background: An adequate knowledge of physiological variation is important for valid comparative studies of tear proteins. The aim of this study was to investigate the between-day variation of the human tear protein levels, including the total protein concentration (TPC) and the levels of major protein fractions. Two sampling methods, the yawn and the eye-flush methods, were used and compared. Methods: TPC was determined by the Bradford method. The major protein fractions were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and their levels were determined by scanning densitometry after SDS-PAGE. The tear protein levels were monitored for 3 days. Results: The between-day differences in the levels of TPC and the individual protein fractions were not statistically significant in either sampling method, but the variations of some proteins were large and would be clinically significant. Different variations were observed in different proteins. The variations in serum albumin were large, up to 61% and 113% in the yawn and eye-flush methods respectively. The variations in lactoferrin, tear-specific prealbumin and lysozyme were relatively small in the yawn method. The variations in protein levels obtained by the eye-flush method were generally higher than by the yawn method. Conclusion: Although the between-day differences in tear protein levels were not statistically significant, the variations in some proteins would be large in magnitude. The variability of tear protein levels obtained by the eye-flush method was larger than that by the yawn method. Therefore, caution should be taken if the eye-flush method is used for sampling tears for quantitative analysis of tear proteins, although it is easier to collect tear samples using this method. The results will be useful to exclude normal variation in tear protein levels when comparing pre- and post-therapeutic tear protein levels in eyes treated for tear-related abnormalities. Received: 22 March 2000 Accepted: 19 April 2000     

新生儿眼部感染情况分析

目的:研究2014/2018年浙江苍南县新生儿眼部感染病原菌分布、常用抗生素耐药性及流行特征,为临床病情的预防、诊断与治疗提供依据。方法:将本院2014-01/2018-12新生儿门诊294份眼部标本中分离所得108株病原菌进行鉴定、常用药物敏感试验和流行病特征汇总分析。结果:眼部分泌物标本294份中病原菌检出率为36.7%(108/294)。主要菌群包括表皮葡萄球菌48株(44.4%),金黄色葡萄球菌16株(14.8%),大肠埃希菌24株(22.2%),淋病奈瑟菌12株(11.1%),类白喉棒状杆菌5株(4.6%),肠球菌属2株(1.9%),链球菌属1株(0.9%)。主要病原菌对阿米卡星和奈替米星具有高度敏感性,对洁霉素和庆大霉素活性较差。对喹诺酮类抗生素活性较好。结论:新生儿眼部感染的致病菌以葡萄球菌属为主,临床常用眼部抗菌药物对各类病原菌具有良好的抗菌活性。     

应用非接触性眼表综合分析仪观察T2DM和/或高血压患者的眼表特征

目的：使用非接触性眼表综合分析仪分析2型糖尿病(T2DM)和/或高血压患者眼表特征。

方法：选取2017-01/2018-12在我院就诊的T2DM患者、高血压患者、T2DM合并高血压患者、无T2DM也无高血压患者各40例40眼,分析比较四组患者OSDI评分、泪河高度、眼红指数、角膜荧光素钠染色评分、非侵入性泪膜破裂时间(首次泪膜破裂时间和平均泪膜破裂时间)检测结果。

结果：T2DM患者眼部不适的主观感受更为明显,T2DM和/高血压患者更容易出现眼部充血的体征。与无T2DM也无高血压的患者相比,T2DM患者的泪河高度降低,平均泪膜破裂时间明显缩短(均P<0.05),但四组患者的首次泪膜破裂时间、角膜荧光素钠染色评分均无明显差异(P>0.05)。

结论：非接触性眼表综合分析仪能够无侵入地观察眼表特征,提供相对客观的量化检查结果。T2DM患者眼部稳定性较无T2DM也无高血压的患者下降,稳态更容易被打破。     

Antimicrobial peptides: a potential role in ocular therapy

Bacterial pathogens are often involved in contact lens‐related adverse responses. This study aimed to find antimicrobial peptides and proteins that effectively eradicate or inhibit ocular bacteria. The antimicrobials were screened against Gram‐ negative and Gram‐positive bacteria originating from ocular sources. The viability of these ocular bacteria was measured after exposure to the peptides and proteins. Two conditions were used to grow bacteria, low nutrient phosphate‐buffered saline and high nutrient tryptone soya broth. Samples were taken at different times up to 48 h. In low nutrient conditions, protamine was found to be the most effective against all strains. Melittin was very effective against all strains except Serratia and one Pseudomonas isolate which were partially affected. In high nutrient condition, only melittin was effective in killing Staphylococcus aureus. Protamine and the combination of protamine and melittin had the greatest effect in eradicating the bacteria tested in low nutrient condition. Protamine alone and its combination with melittin may have potential therapeutic agents for ocular infections in an era of emerging antibiotic resistance.     

超声乳化白内障摘除联合小梁切除术对眼表的影响

目的：采用Keratograph 5M眼表综合分析仪比较小梁切除术和超声乳化白内障摘除联合小梁切除术对眼表的影响。

方法：纳入原发性闭角型青光眼合并白内障患者62例62眼,按手术方式分为两组：小梁切除术组32例32眼,超声乳化白内障摘除联合小梁切除术组(青白联合手术组)30例30眼。运用Keratograph 5M评估术前,术后3d,1、 3mo的非侵入性首次泪膜破裂时间(NifBUT)、非侵入性平均泪膜破裂时间(NiaBUT)、泪河高度(TMH)和角膜荧光素染色评分(CFS)。

结果：术前两组患者眼表参数比较差异均无统计学意义(P>0.05)。术后3d青白联合手术组的NiaBUT、NifBUT、CFS、TMH最差,分别为10.13±1.48、12.59±1.96s、0.80±0.22分与0.31±0.02mm,变化幅度明显高于小梁切除组(均 P<0.05),术后1mo两组的各项指标均有所恢复,但直到术后3mo仍未完全恢复到术前水平。

结论：眼表综合分析仪可以客观、精确地用于评估抗青光眼手术后泪膜功能的变化。在术后3mo短期内超声乳化白内障摘除联合小梁切除术比单纯小梁切除术对眼表的影响更为严重,提示在此期间应加强对眼表的护理。     

口服降糖药和胰岛素对眼表治疗的影响

目的:描述和对比口服降糖药(OAD)和胰岛素治疗2型糖尿病患者眼表的效果。方法:在安卡拉卫生部培训和研究医院就诊的20例40眼口服降糖药患者,20例40眼胰岛素治疗患者,对照组的10例非糖尿病人作为研究对象。对所有患者进行常规眼科检查,Schirmer试验,泪膜破裂时间分析和结膜印迹细胞学分析.比较三组的泪液功能参数,杯状细胞密度和鳞状上皮化生等级。记录视网膜病变及眼表疾病之间的关系。结果:口服降糖药患者泪膜破裂时间值明显低于其他组(P<0.05)。三组的杯状细胞密度和鳞状上皮化生等级相近。上皮细胞稍大,呈现更多的多边形的眼的中位数级为1级,并且嗜酸性染色胞浆和杯状细胞数量减少。视网膜病变与眼表疾病不相关。结论:口服降糖药患者的角膜前泪膜稳定性比其他组差。各组的印迹细胞学分析结果相似。     

Ocular pathogen or commensal: a PCR-based study of surface bacterial flora in normal and dry eyes

PURPOSE: To compare the bacterial population of the ocular surface of normal and dry eye subjects using conventional culture and 16S rDNA PCR. METHODS: Ninety-one subjects were classified as normal (n = 57) or dry eye (n = 34) by using tear break-up time, McMonnies survey, goblet cell density, and meibomian gland assessment. Conventional bacterial culture and broad-range 16S rDNA PCR, cloning, and DNA sequencing were used for bacterial identification. Repeated sampling was performed in a subset of subjects over a 3-month period. The association between goblet cell loss and bacterial counts in a subgroup of subjects was assessed. RESULTS: Most of the bacteria identified by culture were coagulase negative staphylococci, whereas molecular methods demonstrated a considerable number of additional bacteria. Atypical ocular surface bacteria including Rhodococcus erythropolis, Klebsiella oxytoca, and Erwinia sp., were identified in cases of overt inflammation and, surprisingly, on the normal ocular surface. The same bacteria remained on the ocular surface after repeated sampling. Increased bacterial flora was associated with reduced goblet cell density. CONCLUSIONS: Molecular analysis revealed a diverse ocular surface bacterial population. In addition to the normal flora, various potentially pathogenic bacteria were identified. The detection of known pathogens in both normal and dry eyes, with minimal signs of infection, presents a diagnostic dilemma. It remains unknown whether their presence is associated with inflammation and reduced goblet cell density or whether they adversely affect the ocular surface predisposing it to abnormal microbial colonization. In the absence of overt clinical infection, it is unknown whether such results should prompt intervention with therapy.     

翼状胬肉患者眼表微生物菌群分析

目的：研究翼状胬肉患者的眼表微生物菌群组成,探索眼表微生物菌群和翼状胬肉的相关性。

方法：收集2018-09/2019-01在我院就诊的原发性翼状胬肉患者,翼状胬肉组为患翼状胬肉的26眼,正常对照组为9只对侧正常眼,采用MiseqPE301+8+8+301平台进行细菌16S rDNA基因V3～V4区双端测序,分析比较翼状胬肉组与正常对照组眼表微生物菌群结构与组成差异。

结果：两组样品共得到1 837个OTU; Alpha多样性分析,正常对照组物种的多样性高于翼状胬肉组。同时两组优势菌群相似。构成以棒状杆菌、葡萄球菌、链球菌、丙酸杆菌等革兰氏阳性菌为主,其次为革兰氏阴性菌。棒状杆菌在翼状胬肉组比例增高。

结论：翼状胬肉组眼表物种多样性下降,棒状杆菌在翼状胬肉患者眼表菌群的丰度显著增高,可能参与眼表免疫状态调节,并影响翼状胬肉的发生与发展。     

Retinal pigment epithelial tear and extensive exudative retinal detachment following blunt trauma

Background: A peripheral retinal pigment epithelial tear and an extensive exudative retinal detachment caused by choroidal leakage from the denuded Bruch’s membrane are extremely rare. A peripheral retinal pigment epithelial tear has not been reported in an eye with retinochoroidal folds after blunt ocular trauma. Methods: Case report. Results: The course of a large nasal peripheral retinal pigment epithelial tear that occurred after blunt ocular trauma in a patient with retinochoroidal folds was followed. The inferior retinal detachment caused by leakage from the denuded Bruch’s membrane following the development of the tear gradually worsened. Initial treatment with cryotherapy was ineffective, but the retinal detachment eventually resolved after the patient underwent sclerectomy and sclerostomy. Conclusion: A large peripheral retinal pigment epithelial tear can occur in patients with retinochoroidal folds following blunt ocular trauma, and extensive retinal detachment can be induced. Sclerectomy and sclerostomy can be beneficial in patients with an extensive exudative retinal detachment caused by choroidal leakage from the denuded Bruch’s membrane. Received: 12 October 1999 Revised: 6 January 2000 Accepted: 9 March 2000