【摘要】目的:分析6种角膜接触镜多功能护理液和加入甲硝唑滴眼液的护理液对自生生活性棘阿米巴原虫的杀伤效果。方法:将6种多功能护理液分别加入96孔板中，每种护理液占用48孔，其中24孔滴入阿米巴悬液，另外24孔先滴入甲硝唑滴眼液后再滴入阿米巴悬液，室温静置8h后在倒置显微镜下观察残存阿米巴的形态变化和数量。将残存的棘阿米巴原虫分别在PYG培养液中培养5d，观察其形态、活性与增殖能力的变化。结果：单纯护理液组16号阿米巴检出率分别为0%、80。3%、29.1%、41.7%、62.5%、79.2%，加入甲硝唑滴眼液后护理液16号阿米巴检出率分别为0%、0%、4.2%、8.3%、16.7%、16.7%，36号护理液加与不加甲硝唑滴眼液杀伤阿米巴的效力差异有统计学意义（36组的χ2值分别为3.75、7.11、10.54和18.78，P<0.05）。残存的棘阿米巴原虫经培养后活力与增殖力减弱。结论:部分多功能护理液对棘阿米巴原虫的杀伤效果不佳，添加甲硝唑滴眼液后杀伤效果明显提高
【关键词】 棘阿米巴；接触镜护理液；甲硝唑
The killing efficiency of arilin and contact lens solutions on Acanthamoeba cultured in vitro
ChengYe Che, GuiQiu Zhao, LiLi Zhang
Department of Ophthalmology, the Affiliated Hospital, Medical College of Qingdao University, Qingdao 266003, Shandong Province, China
Correspondence to: GuiQiu Zhao. Department of Ophthalmology, the Affiliated Hospital, Medical College of Qingdao University, Qingdao 266003, Shandong Province, China. [email protected]
AbstractAIM: To analyze the killing efficiency of six kinds of contact lens solutions and solutions with arilin on freeliving Acanthamoeba cultured in vitro.METHODS: Six kinds of contact lens solutions were added into 96well microtiter plates, respectively, with each care solutions used 48 holes of them. Suspension of Acanthamoeba were added into 24 of these holes, and arilin gutta and suspension of Acanthamoeba were added into the other 24 holes. After standing in room temperature for 8 hours, the morphologic change and quantity of the remnant Acanthamoeba were observed under the inverted microscope. The remnant Acanthamoeba were cultivanted in peptoneyeast extractglucose (PYG)culture medium for 5 days. Their variation of appearance, activity and reproductive activity were observed.RESULTS: In the six experimental groups using contact lens solutions only, the detection rate of Acanthamoeba of were 0%, 80.3%, 29.1%, 41.7%, 62.5% and 79.2%, respectively. After arilin was added, the detection rate of Acanthamoeba of the six groups were 0%, 0%, 4.2%, 8。3%, 16.7% and 16.7%, respectively. From group 3 to group 6, after arilin was added, the differences of the killing efficiency of contact lens solutions have statistical significance (χ2=3.75, 7.11, 10.54 and 18.78; P<0.05). Cultivation of the remnant Acanthamoeba showed a reduction in the activity and proliferative ability.CONCLUSION: The killing efficiency of some contact lens solutions on freeliving Acanthamoeba were not satisfying. Arilin can improve the killing efficiency of contact lens solutions.
KEYWORDS: Acanthamoeba castellanii; contact lens solutions; arilin
INTRODUCTION
Acanthamoeba keratitis is a severe sightthreatening corneal disease caused by Acanthamoeba castellanii protozoa. It has been confirmed overseas that corneal abrasion and wearing amebaladen contact lenses are the primary mechanism for the provocation of this disease in humans [1]. The qualified contact lens solutions should have three functions: depuratory, degermation and deproteinization [2]. So, it ought to play an important role in the precaution of Acanthamoeba keratitis. In our experiment, six kinds of contact lens solutions in the market were chosen to be cultured with freeliving Acanthamoeba in order to inspect their killing ability. Then we compared the killing ability of the former with their corresponding contact lens solutions added with arilin gutta.
MATERIALS AND METHODS
Cultivation of Acanthamoeba Castellanii Standard Acanthamoeba castellanii (CW1) were purchased from Beijing Institute of Ophthalmology (Beijing, China). Acanthamoeba castellanii were axenically cultured in a proteose peptoneyeast extractglucose (PYG) medium containing penicillin (500U/mL) and streptomycin (500U/mL), at 28℃ in incubator.
Trophozoites were harvested at the logarithmic growth phase (35 days). Observation under inverted microscope revealed that amoeba had two appearancesin PYG medium: Trophozoites (>80%) and cyst. Trophozoites were irregular round with some weeny processus spinosus in their body surface. The cysts characteristically showed doublewalled structures and the endocysts showed irregular polygons. The number of amoeba was counted with blood cell counting plate. Its concentration was expressed by organisms/mL. The suspension of amoeba was concocted into 3×104 organisms/mL, and then was mixed.
Contact lens Solutions Six kinds of contact lens solutions sold in the market used in our experiment were numbered from 1 to 6. None of them includes arilin or their active component. Their producing dates were from 20070401 to 20070831, with guarantee period of two years. Our experiment date was in their guarantee period.
Empirical Method Three pieces of sterile 96well microtiter plates were used in our experiment. Each kind of contact lens solutions used 48 holes. By using microamount sample injector, 100μL care solution and 1μL suspension of Acanthamoeba (3×104 organisms/mL) were added into each hole. Then 100μL 0.2% arilin gutta were added into 24 of the 48 holes. The existence of Acanthamoeba was verified after added into suspension of Acanthamoeba for 8 hours. PN, positive number; NN, negative number microscopically. After standing in room temperature for 8 hours, the morphologic and quantitative change of the surviving Acanthamoeba in the care solutions were observed under inverted microscope.
Cultivation of the Surviving Acanthamoeba The remanent fluid in these sterile 96well microtiter plates from number 1 to number 6 was collected and then centrifugated (2000rpm for 5 minutes) and its supernatant was removed. The remnant was cultivated with 2mL PYGculture medium. Control group was set simultaneously: 24μL suspension of Acanthamoeba (3×104 organisms/mL) was takenand centrifugated and its supernatant was removed with the same method. The remnant was cultivated with 2mL PYGculture medium. The variation of their appearance, activity and reproductive activity were observed under inverted microscope 5 days later.
Statistical Analysis All the data were analyzed with Chisquare test of fourfold table. The detection rates of amoeba of each group in the two experiments were analyzed with paired comparison. P<0.05 showing the difference had statistical significance.
RESULTS
Observation of Acanthamoeba 8 hours later After standing in room temperature for 8 hours, Acanthamoeba was surveyed under inverted microscope. The detection rates of Acanthamoeba in each group are showed in Table 1.Table 1Comparison of the detection rates of Acanthamoeba in six kinds of care solutions (略)
Observation under inverted microscope showed that in the fluid without surviving Acanthamoeba, only the sporadic cell debris could be seen, and no intact trophozoite or cyst was visible; whereas in the fluid with some surviving Acanthamoeba, the number of Acanthamoeba, which were almost all cysts, was small. A small quantity of cell debris was visible.
In our experiment revealed that among the six kinds of contact lens solutions sold in the market, number 1 and number 2 can kill Acanthamoeba effectively. The killing efficiency of the rest care solutions was poor.
Statistical analysis showed that the difference between the detection rates of acanthamoeba of number 1 and number 2 with or without arilin had no statistical significance. The difference between the detection rates of acanthamoeba of the rest with or without arilin had statistical significances (in group 3 to 6, χ23=3.75, χ24=7.11, χ25=10.54, χ26=18.78; P<0.05).
Observation of Acanthamoeba 5 days Later Observation under inverted microscope showed that Acanthamoeba grew wellin the control group. The percentage of trophozoite was no less than 80%. No living Acanthamoeba was visible in number 1 and number 2 care solutions without arilin. In number 3 to 6 without arilin, the number of Acanthamoeba changed a little, and most of them were cyst. No living acanthamoeba was visible in number 1 to number 3 care solutions with arilin. In number 4 to 6 with arilin, the number of Acanthamoeba had no significant increase, and sporadic cysts were visible.
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