棘阿米巴角膜炎的诊断和治疗探讨

目的 探讨棘阿米巴角膜炎的临床与实验室诊断方法,寻找有效滴眼液用以治疗。方法 观察分析２５例棘阿米巴角为感染各阶段的临床表现,通过角膜细胞学检查、阿米巴分离培养、角膜活检及组织病理学检查确诊,检测药物对棘阿米巴的抗原虫作用及临床疗效。结果感染自角膜上皮层开始,进行性侵入基质致盲。细胞泞凶世囊和／或滚养体（８８．９％）。棘阿米巴培养阳性率５７．９％。洗必泰、甲硝唑滴眼液治疗棘阿米巴角膜炎有良效。抗     

棘阿米巴角膜炎研究进展

棘阿米巴角膜炎系棘阿米巴原虫感染引起的一种顽固性、进行性角膜炎,为严重的眼部感染性疾病.棘阿米巴原虫大量存在于自来水、角膜接触镜护理液等中,随着角膜接触镜佩戴人数的增多,该病的发生有迅速增加的趋势.棘阿米巴角膜炎的诊断主要依靠病史、临床症状和实验室诊断,尤其是后者在近年来的研究中进展较快.棘阿米巴角膜炎的治疗主要是药物治疗和手术治疗,其中准分子激光角膜切削术已成为一种新的治疗方法.就棘阿米巴角膜炎的病原学、诊断和治疗方面的研究进展进行综述.     

直接PCR法对感染性棘阿米巴角膜炎的诊断价值

背景棘阿米巴角膜炎的致盲率极高,发病初期易误诊为真菌性角膜炎或病毒性角膜炎,常规的临床诊断方法费时较长,特异性差,极易错过治疗的“时间窗”。常规PCR法检测简单、特异、有效,但由于病变标本取材量少,不易提取组织DNA,限制了其临床应用。目的探讨非提取组织DNA的直接PCR法扩增棘阿米巴虫株18SrRNA保守序列在棘阿米巴角膜炎快速诊断中的可行性。方法从山东省眼科研究所暨青岛眼科医院诊治的10例棘阿米巴角膜炎患者的病变角膜中分离10株棘阿米巴虫株,经鉴定均为T4基因型虫株。用直接PCR法分别扩增棘阿米巴原虫、白念珠菌、绿脓杆菌、I型单纯疱疹病毒以及正常人角膜上皮细胞以确定该方法的特异性。将棘阿米巴原虫稀释至不同的浓度以确定直接PCR法的敏感性。采用SPF级6周龄健康雌性BALB／c小鼠构建棘阿米巴角膜炎动物模型,于感染后第1、3、5、7、10、15天获取角膜组织,分别进行直接PCR、实时定量PCR（real—timePCR）、K0H封片镜检和原虫培养鉴定,比较各种方法的有效性和可行性。结果直接PCR法仅能扩增棘阿米巴DNA,其他病原体DNA均未扩增出;在每个获取的棘阿米巴角膜炎样本中最少可检测到10个棘阿米巴原虫。在棘阿米巴角膜炎小鼠模型中,直接PCR法在感染后第1、3、5、7、10、15天的阳性检出率分别为80．0％、90．0％、80．0％、70．0％、70．0％和50．0％,总阳性率为73．3％,高于原虫培养法的31．7％,差异有统计学意义（P=0．005）;K0H封片镜检的总阳性率为56．7％,real—timePCR法检测的总阳性率为61．7％,均略低于直接PCR法,差异均无统计学意义（P=0．056、0．172）。结论直接PCR法操作简单,在上述4种方法中对棘阿米巴原虫检测的特异性和敏感性最高,能够快速诊断棘阿米巴角膜炎,尤其对于待检标本少而无法提取DNA的患者可首选。     

棘阿米巴原虫对三种角膜接触镜的黏附性比较及简易处理方法的研究

目的:对三种角膜接触镜与棘阿米巴原虫的黏附能力进行比较,观察加用无菌生理盐水反复吹打角膜接触镜表面,对清除接触镜表面棘阿米巴效果的影响。方法:取市售的三种角膜接触镜分为硬性透气性角膜接触镜(RGP)组、亲水性软性接触镜(SCL)组、彩色角膜接触镜组,并分别与棘阿米巴悬液共同培养16h,镜下观察比较棘阿米巴的黏附数量,并将彩色接触镜的有色区域和无色区域进行比较;加用无菌生理盐水反复吹打角膜接触镜表面后,比较接触镜表面残存的棘阿米巴数量。结果:彩色接触镜组的阿米巴黏附数量高于RGP组和SCL组(P<0.05).RGP组和SCL组之间差别无统计学意义(P>0.05)。彩色接触镜组其彩色区域的棘阿米巴的黏附数量高于无色区域(P<0.01).三组接触镜经吸管用无菌生理盐水冲洗后,棘阿米巴的黏附数量均有明显降低(P<0.01)。结论:与RGP组和SCL组的接触镜相比,彩色接触镜更易受到棘阿米巴原虫的黏附;加用无菌生理盐水反复吹打角膜接触镜表面可明显加强对接触镜表面棘阿米巴的清除效果。     

角膜创伤致棘阿米巴感染的临床特征及治疗

目的 分析角膜创伤致棘阿米巴感染的角膜炎临床特征、治疗及琐后。方法 对20例（20眼）棘阿米巴性角膜炎的危险因素、临床表现、实验室榆查．治疗及其结果进行回顾性分析。结果 20例中，学生12例，农民6例。危险因素中包括戴角膜接触镜12例和角膜创伤6例。主要的临床表现为角膜溃疡、弥漫性基质浸润和环形溃疡。实验室检查：角膜刮片细胞学检查和棘阿米巴培养阳性率分别为95．00％（19／20）和60．00％（12／20），其中4眼在共焦显微镜检查下查见包囊。0．02％洗必泰，0．5％新霉素，0．4％甲硝唑滴眼液联合治疗，同时结合1％洗必泰局部烧灼和角膜清创。平均疗程为70天（18～150天）。19例溃疡愈合，形成角膜薄翳或白斑。其中7眼的最佳矫正视力≥0．5。结论 棘阿米巴性角膜炎的早期诊断至关重要，联合应用抗棘阿米巴药物、局部清创术和烧灼术为目前治疗的最佳选择．     

棘阿米巴性角膜炎的临床特点及诊断

目的 分析、总结棘阿米巴性角膜炎的临床特点及各种病原学检查阳性率比较.方法 回顾分析2002年至2011年因棘阿米巴性角膜炎在山东省眼科研究所收集患者的临床资料.结果 10 a间因棘阿米巴性角膜炎在山东省眼科研究所治疗的患者共30例(30眼),均为单眼患病;其中男20眼,女10眼.所有患者(30眼)均因眼红、痛、视力下降就诊;其中28眼(93.3％)首次就诊我院时即诊为棘阿米巴性角膜炎,2眼(6.7％)误诊为病毒性角膜炎,1周后因治疗效果不佳,再次查找病原学病因才确诊为棘阿米巴角膜炎.所有患眼中,23眼(76.7％)有角膜环形基质浸润,5眼(16.7％)伴剧烈疼痛,4眼(13.3％)伴放射状角膜神经炎.所有患者均通过病原学诊断确诊为棘阿米巴性角膜炎,其中27眼(90.0％)经角膜刮片生理盐水湿片检查查见棘阿米巴包囊;17眼(56.7％)经角膜刮片棘阿米巴原虫培养阳性;行共焦显微镜检查的25眼中,22眼(88.0％)共焦显微镜下查见棘阿米巴包囊.结论 角膜环形基质浸润是棘阿米巴性角膜炎的特征性体征.根据病史、体征及相关病原学检查,绝大多数棘阿米巴性角膜炎患者能够及时确诊.相关病原学检查中,角膜溃疡灶刮片生理盐水湿片检查、共焦显微镜检查较棘阿米巴原虫培养阳性率高.     

共焦显微镜在棘阿米巴性角膜炎临床诊断中的应用

目的 :评价共焦显微镜 (confocalmicroscopy)在棘阿米巴性角膜炎 (acanthamoebakeratitis)临床诊断 ,尤其是早期诊断中的应用价值。方法 :对临床拟诊为棘阿米巴性角膜炎患者 2 3例进行共焦显微镜检查 ,并同时行角膜涂片及棘阿米巴原虫培养检查 ,对两种检查方法的结果进行比较。结果 :2 3例患者中 ,13例共焦显微镜检查可见棘阿米巴包囊和 /或神经炎表现 ,综合各项检查结果及临床表现 ,18例确诊为棘阿米巴性角膜炎 ,共焦显微镜的阳性率为 5 6 5 %。在 5例表现为上皮性及浅基质病变的早期角膜炎患者 ,共焦显微镜检查 3例 (6 0 % )为阳性 ,刮片、培养各 1例 (2 0 % )为阳性。结论 :共焦显微镜检查是一种无创、快速和有效的活体检查手段 ,在棘阿米巴性角膜炎的诊断中 ,如同时结合实验室刮片及培养和患者临床情况可起到重要的辅助诊断作用 ,尤其是在棘阿米巴角膜炎的早期诊断中 ,可以起到更重要的作用     

棘阿米巴性角膜炎发病机制及免疫反应研究的进展

棘阿米巴角膜炎的发病机制包括原虫对角膜上皮的黏附、释放蛋白酶非接触性细胞溶解作用、接触性细胞溶解作用以及介导角膜细胞凋亡。机体感染棘阿米巴后产生相应的特异性与非特异性免疫反应,提示有可能对棘阿米巴角膜炎进行预防。     

聚合酶链反应快速诊断棘阿米巴角膜炎的研究

目的:探讨聚合酶链反应(PCR)技术快速诊断棘阿米巴角膜炎的价值。方法:建立棘阿米巴标准虫株的PCR检测方法,并应用于临床检测24例角膜刮片标本,结果与原虫培养及100g/L氢氧化钾湿封片镜检做比较。结果:PCR5h可检测出标本中微量棘阿米巴原虫,对照细菌、真菌、I型单纯疱疹病毒、正常人角膜均为阴性。临床标本PCR敏感性为46%,明显高于原虫培养与100g/L氢氧化钾湿封片镜检(P<0.05)。结论:PCR速度快、敏感性和特异性高,有助于棘阿米巴角膜炎的快速明确诊断。     

兔棘阿米巴角膜炎的组织病理学研究

目的 观察兔棘阿米巴角膜炎角膜组织感染的病理变化及其特点,探讨棘阿米巴角膜炎的发病机制.方法 新西兰白兔20只,其中16只角膜基质内注射纯化培养的棘阿米巴原虫悬液建立兔棘阿米巴角膜炎模型,观察兔角膜组织的病理学及免疫学的相应变化以及与组织降解和修复相关的细胞因子基质金属蛋白酶13(MMP13)及碱性成纤维细胞生长因子2(bFGF2)的表达;4只为对照.结果 兔棘阿米巴角膜炎感染,初期以中性粒细胞浸润为主,21d后以巨噬细胞为主,并伴有成纤维细胞的增生,7d和14d时 CD4+CD8+细胞比值异常;感染初期,组织内开始出现MMP13阳性表达,从14d开始MMP13合成减少,FGF2的表达逐渐增强,28d时达峰值,其后减弱.结论 兔棘阿米巴角膜炎早期的自然病程以感染性炎症为主,后期主要特征为角膜组织修复.MMP13和FGF2的表达变化与临床表现有一定相关.     

Acanthamoeba keratitis and contact lens disinfecting solutions

OBJECTIVES: To report cases of culture-proved Acanthamoeba keratitis in Greece over a 10-year period and to evaluate the effectiveness of the commonly used commercial contact lens disinfecting systems in clinical cases of Acanthamoeba keratitis. MATERIAL AND METHODS: During the years 1994-2004, 45 contact lens wearers and 3 non-contact lens wearers presenting with symptoms and signs of keratitis underwent corneal sampling. The scrapings obtained were inoculated directly onto appropriate culture media for bacteria, fungi and Acanthamoeba. All proved positive for Acanthamoeba. The contact lenses and contact lens disinfecting solutions (16 one-step 3% hydrogen peroxide and 3 multipurpose solutions) of 19/45 patients with culture-proven Acanthamoeba keratitis were cultured for bacteria, fungi and Acanthamoeba. RESULTS: Acanthamoeba was isolated from contact lenses and contact lens disinfecting solutions in all 19 cases of Acanthamoeba keratitis studied. CONCLUSIONS: The main risk factor for corneal infection in contact lens wearers is the use of contact lens disinfecting systems ineffective at killing Acanthamoeba cysts and trophozoites, as well as bacteria and fungi. Improvement or development of new contact lens disinfecting systems by manufacturers is needed to prevent Acanthamoeba keratitis.     

The effect of currently available contact lens disinfection systems on Acanthamoeba castellanii and Acanthamoeba polyphaga

Contact lens disinfection systems were evaluated for their effectiveness in killing Acanthamoeba castellanii and Acanthamoeba polyphaga trophozoites and cysts. Amoebae were inoculated into commercially available contact lens cleaning and soaking solutions. At intervals varying from 30 minutes to 24 hours, solutions were filtered. The filters were removed and cultured for Acanthamoeba organisms. Striking differences were observed in the abilities of the different disinfecting solutions to kill the organisms. Solutions containing chlorhexidine were effective at very short exposure times. Solutions containing benzalkonium chloride required slightly longer exposure times but were faster than solutions containing only thimerosal. Solutions containing sorbate, polyaminopropyl biguanide, or polyquaternium-1 were not effective at killing Acanthamoeba organisms in the time allotted for the experiment. Solutions containing hydrogen peroxide were quite effective if the agent was not prematurely catalyzed. A. polyphaga generally required longer exposure to disinfectants than did A. castellanii for complete inhibition to occur.     

Effectivity of contact lens disinfection systems against Acanthamoeba culbertsoni

Thirteen commercially available contact lens solutions were tested for their ability to kill the cysts of Acanthamoeba culbertsoni. Miraflow, which contains 20% isopropyl alcohol, was the most effective at killing the cyst (94%), followed by solutions containing thimerosal (89%). The rigid gas permeable lens solutions in general were more effective than soft lens solutions. None of the solutions tested were completely cidal, but our data do suggest a combination of a good daily cleaner and disinfecting solution may be effective in reducing acanthamoeba exposure. These findings should provide guidelines for the practitioner in selecting the best disinfection system for the contact lens patient.     

Microwave treatment of contact lens cases contaminated with acanthamoeba.

PURPOSE: Microbially contaminated contact lens cases are a predisposing risk factor for Acanthamoeba keratitis. Several findings have shown that microwave irradiation kills the six Food and Drug Administration test challenge microorganisms. We aimed to determine what effect microwave irradiation has on Acanthamoeba trophozoites and cysts. METHODS: Different types of contact lens cases were contaminated with trophozoites and cysts of three different Acanthamoeba species (A. comandoni, A. castellanii, A. hatchetti) and were exposed to microwave irradiation for 3, 5, and 8 minutes, respectively. RESULTS: Trophozoites, as well as cysts of the different Acanthamoeba strains, were effectively killed, even by only 3 minutes of microwave irradiation, and there were no negative effects of irradiation on the contact lens cases themselves. CONCLUSION: We demonstrate that microwave treatment is a very effective, easy, and cheap method to keep contact lens cases free of Acanthamoeba, thus considerably reducing the risk of an Acanthamoeba keratitis.     

Viability of Acanthamoeba after exposure to a multipurpose disinfecting contact lens solution and two hydrogen peroxide systems

BACKGROUND/AIM: Contact lens cases contaminated with Acanthamoeba are a major risk factor for an infection of the eye. In this study the anti-Acanthamoeba activity of three different contact lens storage solutions was tested. METHODS: A new multipurpose contact lens storage solution (Meni Care Plus) and a two step (Titmus H(2)O(2)) and one step (Oxysept Comfort) hydrogen peroxide system were tested for their effects on trophozoites and cysts of three different Acanthamoeba species: A castellanii, A hatchetti, and A lenticulata. RESULTS: After a soaking time of 8 hours (overnight soaking of contact lenses) the Titmus H(2)O(2) 0.6% solution showed very good amoebicidal effects, while Oxysept Comfort 3% H(2)O(2) could not effectively destroy the cysts of any of the three tested species. Viable cysts of the species A lenticulata and A hatchetti were still present after exposure to Meni Care Plus (0.0005% PHMB) for 8 hours. CONCLUSION: Not all of the three tested contact lens storage solutions have sufficient amoebicidal effects. The two step peroxide system Titmus H(2)O(2) is a very effective disinfectant contact lens solution in order to avoid a possible Acanthamoeba infection of the eye.     

Microbial analysis of contact lens care systems contaminated with Acanthamoeba

We analyzed bacterial and fungal contamination within the contact lens care systems of ten patients who had Acanthamoeba detected within their care systems. Seven patients had Acanthamoeba keratitis, one had Pseudomonas keratitis, and the remaining two were asymptomatic. Gram-negative bacteria were found in all ten care systems, and Pseudomonas was found in six. Bacillus species, the only gram-positive bacteria isolated, were found in five systems. Fungi were isolated in six care systems. The use of homemade saline and the two-cup method of peroxide disinfection were associated with microbial contamination. Acanthamoeba organisms were found only in contact lens cases or solutions that also had bacterial and in many cases fungal contamination, suggesting that the presence of bacterial and fungal contamination within the contact lens care system may be an important element for the survival and growth of Acanthamoeba.     

Microbial contamination of contact lens care systems

We examined the contact lens care systems of 100 asymptomatic patients who used hard or soft contact lenses for correction of refractive errors for the presence of bacteria, fungi, Acanthamoeba, and endotoxin. Of 100 patients, 52 had contaminated contact lens care systems. Sixteen of 126 bottles (13%) of commercial contact lens care solutions were contaminated. Contaminated commercial solutions were opened and used for a longer period of time than uncontaminated solutions. Contamination was not found in bottles of preserved commercial solutions that were opened and used for less than 21 days. All 12 bottles of homemade saline were contaminated with bacteria, and Acanthamoeba was isolated from two of these bottles. Pseudomonas was found in the care systems of 12 patients. Bacillus species, which form spores resistant to heat, were found in the care systems of seven patients. Endotoxin, which is also resistant to heat, was detected in nine of 35 care systems (26%) tested. Potential pathogens were isolated from the conjunctiva of six patients.     

Detachment of trophozoites of Acanthamoeba species from soft contact lenses with BEN22 detergent, BioSoak, and Renu multi-purpose solutions.

PURPOSE: BEN22 detergent was studied for its ability to detach Acanthamoeba from soft contact lenses without mechanical cleaning or separate cleaning agents. METHODS: Trophozoites of Acanthamoeba castellanii and A. polyphaga were adhered onto nonionic, high water content soft contact lenses. The lenses were immersed for 2 hours in contact lens care solutions and the remaining trophozoites were counted microscopically. The counts were compared to the counts on the same lens before treatment. RESULTS: BEN22 (50:50 mixture of L-alpha-L-rhamnopyranosyl-beta-hydroxydecanoyl-beta-hydroxydecanoate and 2-O-alpha-L-rhamnopyranosyl-alpha-L-rhamnopyranosyl-beta-hydroxydecanoyl-beta-hydroxydecanoate) (Kassell Industries, Inc., Wisconsin Dells, WI) in a concentration of 0.05% detached the trophozoites to a statistically significant greater extent than saline, but commercial ReNu Multi-Purpose Solution (Bausch & Lomb, Italy) and BioSoak (Finnsusp Ltd., Finland) did so as well. ReNu Multi-Purpose Solution was more effective than 0.005% BEN22 in detaching the trophozoites of both of the Acanthamoeba strains. After the 2 hour immersion period, a maximum of 97% of the initial trophozoites were detached. The variation between individual lenses was significantly greater than that within the different areas of one lens. CONCLUSIONS: BEN22 had no reliable detaching effect on Acanthamoeba. The variation between lenses was great, and the rate of detachment was low with all the agents tested indicating that immersion and rinsing in the solutions tested cannot be considered as a safe substitute for proper disinfection against Acanthamoeba in contact lens care.     

Comparative antimicrobial efficacy of soft and rigid gas permeable contact lens solutions against Acanthamoeba

Ten soft contact lens cleaners, three rigid gas permeable contact lens (RGP) disinfecting-soaking solutions, and four hydrophilic contact lens disinfecting solutions were evaluated by laboratory challenge procedures for their antimicrobial efficacy against trophozoites and cysts of Acanthamoeba polyphaga and Acanthamoeba castellanii. MiraFlow was the only cleaner that killed trophozoites and cysts on all lenses during the cleaning step. A disinfecting solution preserved with chlorhexidine and one preserved with benzalkonium chloride killed trophozoites of A. polyphaga within 4 hours, but cysts persisted in both solutions for at least 2 days. Cysts of A. polyphaga survived in three disinfecting solutions up to one week, whereas those of A. castellanii were recovered from six disinfecting solutions at 7 days. An RGP solution that is marketed only in Japan was effective against trophozoites and cysts of both species within 30 minutes.