How Effective Iodine Is Against Acanthamoeba

1. ABSTRACT Acanthamoeba species can cause lesions on the skin and eye infections. Acanthamoeba keratis is a form of infection caused by improper disinfectant of contact lens. These organisms lurk in our water supplies preying on bacteria. The disinfectants in our water supply may kill most bacteria, but Acantamoeba are much more resistant. A study was done at Tennessee Technological University in Cookeville, TN to see how effective iodine would be against unknown Acanthamoeba species. After the protists were grown with PCB and washed with Tris Buffer Saline solution, amoeba were added to an iodine concentration solution of .075 μm/l, destroying all amoeba within two hours. This concentration in water supplies would be a safe amount to ingest. This method of cleansing would also apply to contact lens because of its time efficient capabilities. The results of the data show that this disinfectant could be a much better method to use. INTRODUCTION Acanthamoeba are single-celled organisms that are much bigger than bacteria found in soils, dust, and any type of fresh water. They can cause eye infections, skin lesions, and severe sickness symptoms. They can enter the body through cuts of skin, oral and nasal ingestion, or water splashed in eyes. People can develop eye infections, by not properly taking care of their contacts lenses, and they are at higher risk if making their contact lens cleaning solution. Acanthamoeba keratitus is a corneal infection believed to be high risk for contact users (Yang and Cao 1997). It can be extremly painful in eyes. Chlorine is a well-known disinfectant put in water supplies. It may be highly effective for bacteria; however, destroying bacteria in water starves Acantamoeba forcing them to cyst and making them resistant against the toxic effects (Cursons and Brown 1980). About 15 years ago, Brandt and Ware (1989) tested the capabilities of contact lens care solutions and concluded that amoeba cysts were not destroyed in a reasonable time frame. Acanthamoeba prove to be very strong organisms to be able to survive for long periods of time in toxic solutions. Iodine will be used in this study to test the Acanthamoeba’s tolerance. Appendix 1 shows MSDS chart of iodine. Iodine has been used with other disinfectants like Hydrogen peroxide to enhance killing of Acanthamoeba (Hughes and Andrew 2003). To actually see what iodine can do by itself to Acanthamoeba is the main objective. Depending on which concentrations are effective, this disinfectant could have potential use in water supplies. The concentration amounts used in experiment pose no problem for drinking water (Pedersen and Laurberg 1999). A conclusion will be drawn whether this type of disinfectant method will be safe. METHODS AND MATERIALS Experimental research was conducted at Tennessee Technological University in Cookeville, TN. The Acanthamoeba microorganismsand experimental guidance of project were provided by Professor Gunderson (Biology Department, Tennessee Technological University). Procedures used for experiment were similar to Sutherland and Berk (1996). For safety reasons, the unknown species are incapable of causing infection. The amoeba were cultivated using PCB(plate count broth), which consists of 5 g bacto typtone, 2.5 g bacto yeast extract, 1 g bacto dextrose, and 15 g bacto agar for liter with a final pH of 7.0 ± .2 at 25˚C. Six vials were placed in the ventilated over hood with 15 ml of PCB each. Using a pipette, a few drops of the Acanthamoeba were evenly distributed to the growth medium vials. The vials were then incubated for 7 days at a temperature of 25˚C. The growth medium vials were then washed with Osterhout’s/Tris Saline solution. The solution consists of 10.50 g NaCl, 0.23 g KCl, 0.10 g CaCl2, 0.40 g MgSO4۰7H2O, and .85 g MgCl2۰6H2O per liter. It is prepared by filtering it through a .22 µm pore diameter filter and storing at 4 ˚C. Using 40 ml of the solution, 0.121 g of Tris is added with pH adjusted to 7.6-7.8. A drop of the acanthamoeba solution was viewed and self counted with an inverted microscope giving a population roughly around 32,333 per ml. The Acanthamoeba solution was distributed equally to make three rows of six columns of iodine concentrations. A seventh column was set up as a control group. Saturated iodine, which is the right amount of iodine it takes to dissolve, was the solution type used in the first six columns. Using Merck Index, the first vial was .00015% iodine with solubility of .03 g/ 100 ml. The following vials were half the concentration of the previous in line. The amoeba were exposed to the iodine solution for two hours, then a population count was performed. RESULTS For every 50 amoeba observed in each vial, a count was reported of their status defining number alive to the number dead. The Acanthamoeba were tediously counted with naked eye viewing inside microscope and identified by their state of condition. If the amoeba were floating around like logs, they were viewed as being lifeless. It was very easy to distinguish them by their deteriorated membrane appearance. The amoeba still living had pseudopods still attached to bottom of vial or formed cysts. . Since 2 ml of the experimental vials contained the washed Acanthamoeba, each one had a total of 64667 amoeba. To calculate the total population status of amoeba per vial, the ratio of the 50 counted was multiplied by the total number of amoeba in Table 1. Three series of different iodine concentrations were setup between 0.000150 % to 0.000005 %. The controlled group suffered no complications, all appeared fine and healthy. When observing the amoeba exposed to the iodine solutions two hours later, vial one and two had completely killed off the Acanthamoeba shown in table 2. When a concentration of .0038% was used, some survivors started to occur. A significant change in resistance to the iodine concentrations is greatly noticed in figure 1. It is after vial 5 that the number of amoeba alive outweighed the number dead. An average of the 3 series was taken. There was very little difference among the three, except in the sixth vial. The three numbers recorded had some variation. Appendix II to shows raw data. DISCUSSION Using Iodine as a disinfectant for contact cleaning solutions to treat Acanthamoeba keratis is highly favorable. It greatly reduces the risk of eye infections killing other harmful pathogens as well (Yang and Cao 1997). Just after 2 hours, .0075% iodine solution killed the amoeba at maximum level. The task is completed at a more efficient rate compared to other disinfectants (Brandt and Ware 1989). In comparison with chlorine, the amount of Cl- used was 10 times greater than the amount of iodine that effectively killed all amoeba (Cursons and Brown 1980). These results do show increasing interest for use in water supplies. The problem with using chlorine was the amoeba’s ability to form cysts. The halogen would kill the bacteria before the Acanthamoeba, forcing them to starve and causing their membrane to form layers of cysts. This protected it from the toxin. Problems similar with iodine could occur as well. No test with bacteria was done. Not only does the iodine disinfectant work well when combined with others, it is highly effective by itself (Hughes and Andrew 2003). From 9.0 * 10-6 % to 5.0 * 10-6 %, the success of lethal use declines at an extraordinary amount. Smaller concentrations of 9.0 * 10-6 % and above are still very efficient. Any Acanthamoeba that are alive are very unhealthy and may not even have the ability to cause any serious infections due to their condition. If more time be permitted for their exposure, higher death rates would be observed. Recommended iodine concentrations in water are 200 μg/l in Denmark (Pedersen and Laurberg 1999). Not only do the higher iodine concentrations kill the Acanthamoeba effectively, it helps reduce risk of thyroid problems by giving the body recommended concentration. CONCLUSION Finding a method for Acanthamoeba treatment in water supplies was accomplished. The amount that 100% effectively kills the protists also falls into a recommended category for human intake in the body. This could be useful too in fighting eye keratis caused by improper cleansing of contact lens. The time frame it takes to kill the organisms is quite efficient. In the killing process of amoeba, there pseudopods disappear leaving them unable to attach to a surface. Even if the Acanthamoeba were not totally destroyed, their unhealthy condition could cause to just drift in solution. It would be possible to just rinse them off the lens. The only problem that remains with the experiment done is the ability of amoeba cysts. The concentrations could kill bacteria very quickly causing the amoeba to cyst in solutions. This may be a strong barrier for their resistance against the disinfectant. As of now, the experiment proves the hypothesis of Iodine being a great or even better method for the removal of Acanthamoeba. This experiment greatly showed how effective iodine is against Acanthamoeba.. How Effective Iodine Is Against Acanthamoeba Environment: They can inhabit soils, dust, and any type of fresh water, including our drinking water. Infection: Eye infection, lesions on skin, and severe sickness. Accessible through cuts on skin, oral and nasal ingestion, and water splashed in eyes.