细菌感染性眼病病原学分析(1989～1998)

目的：回顾分析眼科培养菌属的分布及其变化。方法：对10年间（1989－1998）培养的4705份标本进行细菌培养阳性率、革兰染色分布、阳性菌属分布及其变化进行分析。结果：培养阳性菌共1339株，平均培养阳性率为28．6％，革兰阳性球菌占55．6％；革兰阳性杆菌占13．1％；革兰阴性球菌占2．8％，革兰阴性杆菌占28．5％，共分离鉴定出50个细菌属种。培养阳性菌中，凝固酶阴性葡萄球菌（主要为表皮葡萄球菌）的比例最高，为25．3％，其次假单孢菌属为18．8％，微球菌属为11．7％，棒状杆菌属为10．1％，金黄色葡萄球菌为8．2％。10年中，革兰阳性球菌占总检出菌的比例随时间呈上升趋势，而革兰阴性杆菌的比例呈下降改变。结论：革兰阳性球菌及革兰阴性杆菌仍为眼部感染的主要致病菌，但前者的比例有逐年增高的趋势，后者呈下降改变，及时了解眼部细菌菌属的分布与变化，对预防与治疗细菌感染性眼病有重要意义。     

丝状真菌所致真菌性角膜炎的临床研究及相关分析

目的 探讨丝状真菌所致真菌性角膜炎的临床研究方法及指导临床治疗的意义。方法 对110例真菌性角膜炎进行10％KOH湿片镜检、大体培养、鉴定培养、药敏试验、病理检查和动物模型实验以指导临床分型及治疗。结果 本组110例中10％KOH湿片镜检可见大分生孢子59例，小分生孢子51例，有隔菌丝101例，无隔菌丝6例，关节状菌丝3例；鉴定培养镰刀菌59株，曲霉菌31株，青霉菌7株，无孢菌5株，头孢子菌2株，链互隔菌2株，附球菌2株，枝顶孢霉菌2株；药敏试验提示氟康唑对丝状菌均呈现耐药性，其他药物对不同的菌属呈现不同的敏感性和耐药性；病理检查可见病变角膜内大量炎细胞及菌丝浸润；15只实验兔角膜划痕接种的动物模型中4例出现角膜真菌感染。结论进行丝状真菌所致真菌性角膜炎的临床研究，对确定真菌在角膜内的生长情况和指导临床分型及选择个性化治疗、减低医疗成本有较大意义。     

河南地区真菌性角膜病致病菌种调查

目的：调查6年间河南地区真菌性角膜病致病菌种。方法：对1996年10月-2002年2月间就诊河南省眼科研究所的真菌性角膜炎患者426人角膜取材行真菌培养，培养阳性的315例标本进一步行致病真菌菌种鉴定。结果：角膜主要致病菌属依次为镰孢菌属（67.9%)和曲菌属(13.0%)。主要致病菌种依次为茄病镰孢菌。梨孢镰孢菌。黄曲霉菌。串珠镰孢菌，互隔交链孢霉和烟曲霉菌。结论：河南地区的角膜首位致病菌属为镰孢菌属，首位致病菌种为茄病镰孢菌。     

人正常发育角膜上皮和角膜基质的凝集素化学研究

目的:研究正常人发育角膜上皮及基质中凝集素亲和糖蛋白的分布及变化,探讨糖类化合物对角膜发育的可能作用。方法:用五种生物素标记的凝集素:麦胚凝集素(Wheat Germ Agglutin WGA)、伴刀豆凝集素(Con-Canavlin A ConA)、花生凝集素(Peanut Agglutin PNA)、蓖麻凝集素(Ricinus Communis Agglutin I RCA-I)扁豆凝集素(Lens Culinaris A LCA)对人正常发育角膜进行凝集素组织化学研究。结果:1.WGA受体主要分布在角膜上皮细胞膜中,尤其在表层上皮细胞中有明显分布,在发育期间胚胎角膜的前部基质中也有WGA受体分布。2.ConA受体分布于角膜上皮细胞膜及细胞质内,在胚胎期角膜基质中有不均一分布。3.RCALCA受体主要分布在角膜上皮基底部细胞膜上。4.PNA受体在胚胎16周角膜上皮细胞中有较少量分布。结论:1.在人正常发育角膜组织中含有ConA、WGA、PNA、LCA、RCA亲和性糖蛋白,这些糖蛋白参与发育角膜构成。2.在人角膜发育成熟过程中,有凝集素亲和糖蛋白的分布变化,这种变化可能与角膜的发育成熟有关。眼科学报1996;12:178～182。     

真菌性角膜炎的病原学分析

目的研究1999～2003年我院真菌性角膜炎的病原学分布。方法回顾性分析我院化脓性角膜炎真菌涂片标本922份、连续性真菌培养标本1814份，并与我院1975～1986、1989～1997年两个阶段比较，结果涂片标本阳性率为27％，(306／922)，与培养的阳性一致率为82％；真菌培养阳性率为25％(456／1814)；上半年与下半年的比例为1：2．2；培养阳性的真菌菌株中，镰孢菌属占34％(155／456)，曲霉菌属占26%，(118／456)；茄病镰孢菌和烟曲霉菌分别是最主要的致病菌株；与我院前两个阶段的资料比较曲霉菌的比例有明显下降而镰孢菌有明显上升的趋势；另外，456份标本中80份伴有细菌培养阳性．结论目前，镰孢菌属和曲霉菌属是广州地区真菌性角膜炎的主要致病真菌。     

细菌性角膜炎病原学及耐药性分析

目的回顾性地分析细菌性角膜炎致病菌属的分布及其耐药性。方法对1985份细菌性角膜炎患者标本进行细菌培养，并分析培养阳性菌属的分布及其耐药性。结果共培养出细菌279株，阳性率为14．06％；革兰阳性球菌、革兰阳性杆菌、革兰阴性杆菌及革兰阴性球菌分别为119株（42．65％）、58株（20．79％）、98株（35．13％）及4株（1．43％）。假单孢菌属比例最高，为20．07％。细菌对氧氟沙星、环丙沙星、左氧氟沙星及妥布霉素的耐药百分比分别为20．2％、35．9％、15．5％及29．4％。老年组对环丙沙星、左氧氟沙星及妥布霉素的耐药百分比明显高于成人组（x^2=14．744，5．835，19．709；P〈0．05）。结论假单孢菌属及葡萄球菌属仍是细菌性角膜炎最常见的致病菌，各种菌属对常用抗生素耐药性均有升高趋势。     

真菌性角膜炎致病菌种属的PCR鉴定

目的 通过对核糖体18S rRNA基因内部转录间隔区(internal transcribed spacer,ITS)序列的PCR鉴定,建立快速、准确地鉴定真菌性角膜炎致病真菌种属的方法.方法 采用眼科常见5种致病菌标准菌株;镜检确诊真菌阳性的角膜炎临床标本29例.其中真菌培养阳性15例,真菌培养阴性14例.所有标本均采用液氮研磨结合小玻璃珠振荡法破碎真菌细胞壁,提取真菌基因组DNA,并以此为模板PCR扩增其18S rRNA基因ITS保守区序列,进行基因测序后在基因库中查询致病菌的种属,与镜检结果比对.结果 禾谷镰刀菌、尖孢镰刀菌、燕麦镰刀菌、烟曲霉、白色念珠菌等5种标准菌株培养物和29例临床真菌样本(包括15例真菌阳性培养物和14例真菌培养阴性的角膜病变组织样本),各5 mg样品中提取的DNA样品的浓度在1.7～22.5 mg·L-1.PCR扩增后5种标准菌株均有目的条带,15例真菌阳性培养物也均有目的条带,14例真菌培养阴性的角膜病变组织样本有2例有目的条带,其余12例没有目的条带.标准菌株的PCR鉴定阳性率100%,镜检确诊真菌阳性的临床样本PCR阳性率58.6%,高于真菌培养法鉴定的阳性率51.7%.PCR产物进行基因测序后在基因库中查出了致病菌的种属,检索结果与镜检结果完全一致.结论 通过核糖体18S rRNA基因ITS序列PCR鉴定真菌性角膜炎致病菌的方法特异性强,灵敏度较传统的培养加镜检法更高.若能改进从角膜病变组织中提取真菌基因组DNA的方法,其灵敏度还能进一步提高.     

眼部致病性不产孢子菌的ITS测序研究

目的：运用真核生物核糖体RNA基因(rDNA)内部转录间隔区(internal transcribed spacer,ITS)测序技术对不产孢子菌进行菌属鉴定,揭示海南岛眼部致病性不产孢子菌的生物多样性。

方法：选取海南眼科医院检验科分离培养并镜检确定为不产孢子菌的真菌菌株19株,所有菌株均分离自感染性眼病患者眼部感染组织,接种于土豆葡萄糖琼脂培养基和沙氏培养基37℃温箱孵育7～21d后,镜检未发现生殖结构。采用玻璃研磨器研磨联合化学法提取真菌DNA,PCR扩增核糖体ITS保守序列,通过GenBank基因数据库对扩增出的ITS序列进行比对分析,确定致病菌种属。

结果：经PCR扩增后19株样本菌均得到清晰的目的条带。基因测序比对结果显示,19株不产孢子菌分属12个种属,包括可可毛色二孢子菌6株,月状弯孢霉1株,节菱孢霉菌2株,neodeightonia subglabosa 2株,红贝菌1株,肉座菌1株,多喙茎点霉1株,红色毛藓菌1株,深海曲霉菌1株,roussoella siamensis 1株,撕裂蜡孔菌1株、茄病镰刀菌1株。

结论：ITS基因测序技术可较准确地鉴定不产孢子菌的种属,是对传统真菌鉴定方法的有益补充。我国海南岛这一热带地区致病性不产孢子菌种属多样,且与多种感染性眼病的发生相关。     

细菌性角膜炎病原学分析

目的：回顾分析细菌性角膜炎致病菌属的分布及变化。方法：对1989－1999年间送检的2220份细菌性角膜炎细菌培养标本进行革兰染色及分类,对不同菌属的分布及变化情况进行回顾性分析。结果：在2220份培养的角膜细菌标本中,培养阳性菌株490 株,阳性率为22．1％。其中革兰阳性球菌250株,占51．0％;革兰阴性球菌2株,占0．4％;革兰阴性杆菌193株,占39．4％;革兰阳性杆菌45株,占9．2％。假单胞菌属的检出率最高,占32．2％,其次为凝固酶阴性葡萄球菌属为18．6％,第3位肺炎球菌12．0％。10年中,革兰阳性球菌检出率呈上升趋势,革兰阴性杆菌检出率则呈下降趋势。结论：假单胞菌属及凝固酶阴性葡萄球菌属仍是细菌角膜炎较常见的致病菌,革兰阴性杆菌检出率下降与革兰阳性球菌检出率升高趋势应引起临床眼科医师的注意。     

角膜致病性丝状真菌基因分型与临床意义分析

目的研究丝状真菌性角膜炎致病菌株的基因分型及临床意义。方法收集北京同仁医院眼科中心2006年1—12月经病原学检查诊断为丝状真菌性角膜炎的患者52例,对分离的致病菌株进行rDNAITS基因分型和形态学鉴定与分析,并对患者进行治疗和随访,分析基因分型与临床多因素的关系。结果基因分型与形态学鉴定种的一致率达90．3％。根据rDNAITS基因的亲缘关系,将52株丝状真菌分为4组：第1组22株（20株茄病镰刀菌和2株尖孢镰刀菌）;第2组12株（8株串珠镰刀菌、3株层生镰刀菌和1株雪腐镰刀菌）;第3组5株（4株少见眼部真菌和1株串珠镰刀菌）;第4组13株（10株曲霉菌属和3株链隔孢属）。第1组、第2组与第3组、第4组相比,患者发病时间、诱发因素、溃疡严重程度、抗真菌治疗的疗效比较,差异均有统计学意义（P=0．000,P=0．03,P=0．01,P=0．035）。结论rDNAITS基因分型能够准确地鉴定角膜丝状真菌,对于少见或鉴定困难的菌株具有明显的优势。由于菌株的基因型不同,丝状真菌性角膜炎的临床特点具有一定的差异。     

Fungal keratitis:84 cases report in Southern Pakistan

AIM: To identify the predisposing factors, causative fungi and to improve the facilities for the laboratory diagnosis of fungal keratitis. METHODS: Two hundred and forty eyes of 240 patients of clinically suspected microbial corneal ulcer were included in the study. Data was collected through history and slit lamp examination. Using standard techniques, corneal scraping was performed.A portion of each scraping was examined by direct microscopy for the presence of fungi, bacteria and acanthamoeba by using 100g/L potassium hydroxide and also by Gram and Giemsa stainings. Another portion was inoculated directly on the surface of solid media such as blood agar, Mac-Conkey agar, chocolate agar and Sabouraud agar in C-shaped streaks for culture. RESULTS: A total of 240 patients with suppurative corneal ulcer were enrolled in the study, out of which fungi were identified in 84 (35%) patients. Of these, 48 (57.1%) were males and 36 (42.9%) females. The age ranged between 22-80 years. Sixty patients (71.4%) belonged to rural population and twenty four (28.6%) to urban population. Trauma with vegetative material was the most frequent cause noted in eighteen (21.4%) patients. Peak incidence was in the months of October-November. Out of 84 eyes with fungal keratitis, fungi alone were the etiologic agents in 74 (80.10%) cases and bacteria with fungi were identified in 10 (11.90%). The most frequently isolated organism was Candida albicans which was found in 66 (78.6%) patients. CONCLUSION: Fungal keratitis is the leading cause of infective corneal ulcer and Candida albicans being the most commonly isolated pathogen in the patients belonging to Southern Pakistan. The direct microscopic examination with potassium hydroxide 100g/L method is a simple, rapid, inexpensive and reliable method in the diagnosis of this infection.     

巴基斯坦南部84例真菌性角膜炎的研究

目的:确定真菌性角膜炎的易感因素、致病真菌,以及提高相应的实验室诊断能力。方法:240例240眼临床疑似微生物感染的角膜溃疡患者进入本研究。通过病史回溯及裂隙灯检查获得病例资料。采用标准技术完成角膜刮片检查。取部分刮片样本加入10%的氢氧化钾液后,以及采用革兰氏和吉姆萨染色,在直接在显微镜下观察是否存在真菌、细菌和棘阿米巴。另一部分刮片样本直接接种在血琼脂、麦康基琼脂、巧克力琼脂和萨布罗琼脂表面。结果:在纳入本研究的240例伴有脓性分泌物的角膜溃疡患者中,84例(35%)被确认为真菌感染。其中48例(57.1%)为男性,其余36例(42.9%)为女性。年龄分布从22-80岁。其中60例(71.4%)为农村患者,其余24例(28.6%)为城市居民。18例(21.4%)曾有植物外伤史。病变高发于10月至11月间。在84例确诊为真菌性角膜炎的患眼,致病原因为单纯真菌者74眼(80.10%),其余10眼(11.90%)为真菌合并细菌感染。分离得到的最常见致病微生物为白色念珠菌,在66眼(78.6%)中被发现。结论:在巴基斯坦南部,感染性角膜溃疡中,最常见的是真菌性角膜炎,而白色念珠菌是最常见的致病原。采用加入10%的氢氧化钾液后直接显微镜下观察是此类感染原简单、迅速、价廉及可信的诊断方法。     

Pathological and electrophysiological features of a canine cone-rod dystrophy in the miniature longhaired dachshund

PURPOSE: To characterize the electrophysiological and histopathological features of a retinal degenerative disease in a colony of miniature longhaired dachshunds known to have a form of progressive retinal atrophy (PRA). METHODS: Serial electroretinograms were recorded from affected homozygous (n = 36) and heterozygous (n = 15) dogs. Morphologic investigations including immunohistochemistry and lectin histochemistry were performed on selected homozygous animals (n = 15). RESULTS: Clinical findings included loss of tapetal hyperreflectivity. The mode of inheritance was autosomal recessive. An early dramatic reduction of cone-specific ERG amplitude with a more modest reduction in rod b-wave amplitude was demonstrated. Progressively, rod specific responses diminished until there were no recordable responses to the ERG stimuli at 40 weeks of age. Morphologic changes confirmed early cone inner and outer segment loss. Other abnormalities included opsin mislocalization and outer nuclear layer thinning due to the subsequent loss of rod photoreceptors. CONCLUSIONS: A novel canine cone-rod dystrophy has been identified.     

Morphological, histochemical and X-ray microanalytical examination of deposits on soft contact lenses in extended wearing.

The deposits on 29 contact lenses of various water content from 20 wearers were analysed by the methods given in the title. Six lenses were used as bandage lenses, the remaining for optical correction. The age of the patients varied from 7 to 56 years, two-thirds being under 45 years. Wearing time had been from 1 week to 1 year, with an average of 13 weeks. The results obtained by the methods applied showed that calcium was present in just over two-thirds of the cases (20/29), other elements being infrequent. Mucopolysaccharides were found in just under two-thirds (18/29). Chlorine was present in one-fifth of the cases. No significant amounts of lipid were detected. Fungi were found in three cases (3/29). Bacteria were also found in these cases, but never without fungi. Evaluation of the methods applied showed that the methods of choice were macroscopical examination and scanning microscopy in combination with X-ray microanalysis, in a few cases combined with histochemistry. None of the methods applied is sufficient for protein analysis.     

Keratomycosis: clinical and microbiologic experience with dematiaceous fungi

OBJECTIVE: To assess the significance of dematiaceous fungi in the causation of keratomycosis. DESIGN: Retrospective, noncomparative case series. PARTICIPANTS: Eighty-eight cases of dematiaceous fungal keratitis seen at the L. V. Prasad Eye Institute, Hyderabad, India from January 1991 through December 1996. INTERVENTION: Only culture-proven cases were analyzed. MAIN OUTCOME MEASURES: Predisposing factors, clinical characteristics, microbiology, treatment methods, and outcome. RESULTS: Of 557 cases of fungal keratitis seen during the study period, dematiaceous fungi were the etiologic agents in 88 (15.7%), after Fusarium in 210 (37.6%) and Aspergillus species in 170 cases (30.4%), respectively. Trauma was the most common predisposing factor (47.7%). Fifty-three eyes (61.3%) had the classical clinical picture of yellow-white, dry raised infiltrate with feathery hyphate edges at initial examination. The characteristic macroscopic pigmentation was seen in only 24 eyes (27.27%). Septate branching fungal filaments were identified in 78 smears (88.63%) on light microscopy, of which 5 (5.7%) also showed the presence of bacteria. Curvularia species dominated the spectrum (22.7%). Treatment was started in 48 eyes with topical antifungal agents, whereas 37 received both oral and topical antifungal agents. Outcome data were available for 68 cases. Forty-nine (72%) responded to medical therapy, whereas 13 eyes required therapeutic penetrating keratoplasty and 6 eyes had to be eviscerated. CONCLUSIONS: This is the largest series of keratitis caused by dematiaceous fungi reported to date. It clearly brings out the clinical importance of this group of corneal infections.     

Senescence-associated beta-galactosidase histochemistry for the primate eye.

PURPOSE: To develop a senescence-associated beta-galactosidase histochemistry and bleaching protocol for the primate posterior pole. METHODS: Rhesus monkey eyes of different ages were enucleated after death, fixed in 4% paraformaldehyde for up to 16 hours, and cryoprotected using a graded sucrose infiltration technique. Ten-micrometer tissue sections were treated with beta-galactosidase, pH 4 (lysosomal) or pH 6 (senescence-associated) activity, for various times. Bleaching of retinal pigment epithelial (RPE) cell and choroidal melanocyte pigment was performed after beta-galactosidase histochemistry using 0.1% to 1% potassium permanganate incubation for 1 minute to 2 hours followed by 0.5% oxalic acid immersion. RESULTS: A 6-hour incubation with beta-galactosidase, pH 4 or 6, demonstrated optimal staining of the RPE. Uniform staining of the RPE for pH 4 beta-galactosidase was seen in both young and old eyes. In contrast, senescence-associated beta-galactosidase (pH 6) staining was seen in the RPE of 16 and 29-year-old, but not 1- and 2-year-old eyes. Senescence-associated beta-galactosidase staining was evident in RPE cells adjacent to cuticular drusen. Optimal bleaching without loss of beta-galactosidase staining was obtained using a 25-minute incubation with 0.05% permanganate. CONCLUSIONS: The senescence-associated beta-galactosidase histochemistry assay, adapted for use in the primate posterior pole, showed staining of RPE cells in older eyes. Visualization of beta-galactosidase activity in the RPE was enhanced by permanganate bleaching of melanin pigment. This technique could be valuable for identifying senescent RPE cells in human eyes.     

真菌性眼内炎44例的致病菌分析

目的:分析真菌性眼内炎病原菌种类。方法:对明确诊断的44例真菌性眼内炎患者的致病菌和其治疗及转归进行总结分析。结果:涂片及培养均为阳性27例,仅涂片阳性17例。致病菌以烟曲霉菌及酵母样真菌为主,合并细菌感染3例,两种真菌并存1例。行玻璃体切割术27例,术后病情好转或稳定18例。结论:真菌性眼内炎致病菌以烟曲霉菌及酵母样真菌为主,烟曲霉菌愈后差,酵母样真菌愈后好,玻璃体切割治疗有效。     

Oval lipid corneal opacities in beagles: VI. Quantitation of excess stromal cholesterol and phospholipid

We identified and quantitated the following lipids in corneas of normolipemic beagles that had oval corneal opacities: total lipids, cholesterol esters, free cholesterol, total phospholipids, phosphatidyl choline, phosphatidyl ethanolamine, sphingomyelin and total triacylglycerols. All except the total triacylglycerols were significantly higher in the 20 affected corneas than in ten normal age and sex-matched controls (P less than 0.005 for each). Few long-chain unsaturated fatty acids were detected. This lipid analysis and the previously reported natural history, ultrastructure, and histochemistry indicate that these opacities result from a primary disorder of corneal lipid metabolism that closely resembles central crystalline corneal dystrophy in man.     

Fungal spectrum identified by a new slide culture and in vitro drug susceptibility using Etest in fungal keratitis

PURPOSE: To investigate the spectrum of fungal species causing keratitis and to test antifungal drug susceptibility to each isolate using Etest. METHODS: Microbial cultures were performed for patients who were clinically diagnosed with fungal keratitis between September 2002 and July 2004. Modified slide culture was established to identify the fungal species of the isolates. Etest (AB BIODISK, Solna, Sweden) was applied to determine the antifungal agent susceptibility of each isolate to itraconazole, fluconazole, and amphotericin B in vitro, respectively. RESULTS: Among 73 eyes of 73 patients with clinical diagnosis of fungal keratitis, 61 strains of fungi were isolated from 61 eyes. The rate of positive culture was 81.3% of all cases. The spectrum of fungal species involved: 58 (95.1%) isolates of filamentous fungi, including the two most common genera-Fusarium (n = 33, 54.1%) and Aspergillus (n = 9, 14.8%),-followed by 16 (26.2%) isolates of other genera of filamentous fungi such as Alternaria (n = 3, 4.9%), Trichophyton (n = 3, 4.9%), Curvularia (n = 2, 3.3%), Chrysosporium (n = 2, 3.3%), Acremonium (n = 2, 3.3%), and Scedosporium (n = 1, 1.6%), 1 (1.6%) yeast of Candida, as well as two (3.3%) dimorphic fungi of Blastomyces and Sporothrix isolate each. Three filamentous fungi of the isolates failed to be identified according to the information provided by slide culture. The results of Etest showed that 20 (60.6%) isolates of Fusarium were susceptible to amphotericin B, whereas all of them were resistant to itraconazole and fluconazole. All nine (100%) isolates of Aspergillus were sensitive to itraconazole, whereas four (44.4%) of them were sensitive to amphotericin B, and only two (22.2%) of them were sensitive to fluconazole. Seventeen (89.5%), 13 (68.4%), and 10 (52.6%) isolates of the remaining 19 organisms were sensitive to amphotericin B, itraconazole, and fluconazole, respectively. CONCLUSIONS: Fusarium and Aspergillus are the most frequent pathogenic organisms in causing fungal keratitis, whereas other species of fungi can also cause corneal infection. In vitro Etest for assessing antifungal drug susceptibility is a simple and practical method and may provide referential information for clinical consideration of choosing antifungal agents to treat fungal keratitis.