L R Saline Saline Solution Solution Health Effects Support Document for Acanthamoeba Health Effects Support Document for Acanthamoeba U. S. Environmental Protection Agency Office of Water ( 4304T) Health and Ecological Criteria Division Washington, DC 20460 www. epa. gov/ safewater/ ccl/ pdf/ acanthamoeba. pdf EPA 822 R 03 012 May 2003 Health Effects Support Document for Acanthamoeba i FOREWORD The Safe Drinking Water Act ( SDWA), as amended in 1996, requires the Administrator of the Environmental Protection Agency to establish a list of contaminants to aid the agency in regulatory priority setting for the drinking water program. In addition, SDWA requires EPA to make regulatory determinations for no fewer than five contaminants by August 2001. The criteria used to determine whether or not to regulate a chemical on the CCL are the following:

° The contaminant may have an adverse effect on the health of persons.

° The contaminant is known to occur, or there is a substantial likelihood that the contaminant will occur, in public water systems with a frequency and at levels of public health concern.

° In the sole judgment of the administrator, regulation of such contaminant presents a meaningful opportunity for health risk reduction for persons served by public water systems.

The Agency's findings for all three criteria are used in making a determination to regulate a contaminant. The Agency may determine that there is no need for regulation when a contaminant fails to meet one of the criteria. The decision not to regulate is considered a final agency action and is subject to judicial review.

This document provides the health effects basis for the regulatory determination for Acanthamoeba. Health Effects Support Document for Acanthamoeba ii ACKNOWLEDGMENTS The Health Effects Support Document for Acanthamoeba, EPA 822 R 03 012, was written by:

Nena Nwachuku, Ph. D., Office of Science and Technology, Office of Water, and Charles P. Gerba, Ph. D., University of Arizona, Tucson, Arizona. The Lead U. S. EPA Scientist on Acanthamoeba is Nena Nwachuku, Ph. D., Health and Ecological Criteria Division, Office of Science and Technology, Office of Water.

Peer review comments on two earlier versions of this document were provided by the following internal EPA peer reviewers:

Rita Schoeny, Ph. D. ( Office of Science and Technology, Office of Water); Paul S. Berger, Ph. D.; Guy Carruthers; David Soderberg; James Sinclair, Ph. D. ( Office of Ground Water and Drinking Water, Office of Water); and Al Dufour, Ph. D. ( Office of Research and Development).

This final version also addresses comments by six external expert reviewers:

Govinda Visvesvara, Ph. D., and Hercules Moura, Ph. D., Centers For Disease Control and Prevention; A. Julio Martinez, M. D., University of Pittsburgh; Walter Jakubowski, WaltJay Consulting; Hassan Alizadeh, Ph. D., University of Texas Medical Center; and Jerry Niederkorn, Ph. D., University of Texas Medical Center.

Management support was provided by Geoffrey Grubbs, Director, Office of Science and Technology, Office of Water, U. S. EPA. Health Effects Support Document for Acanthamoeba iii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi GLOSSARY OF TERMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii 1.0 EXECUTIVE SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 2.0 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 3.0 GENERAL INFORMATION AND PROPERTIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1 3.1 History and Taxonomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1 3.2 General Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2 3.3 Methods of Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 5 3.4 Cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 5 3.5 Significance of Endosymbiosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 5 4.0 OCCURRENCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1 4.1 Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2 4.1.1 Surface Waters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2 <IP_ADDRESS> Freshwaters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2 <IP_ADDRESS> Seawater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2 4.1.2 Tapwater and Bottled Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3 4.1.3 Swimming Pools and Spas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 4 4.1.4 Sewage and Biosolids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 4 4.2 Animal Wastes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 5 4.3 Air, Dust and Soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 5 4.4 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 5 5.0 HEALTH EFFECTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1 5.1 Eye Infections ( Acanthamoebic Keratitis) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 3 5.1.1 Symptoms of Acanthamoeba Keratitis . . . . . . . . . . . . . . . . . . . . . . . . . 5 5 5.1.2 Diagnosis of Acanthamoeba Keratitis . . . . . . . . . . . . . . . . . . . . . . . . . . 5 6 5.1.3 Identification Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 6 5.1.4 Treatment of Acanthamoebic Keratitis . . . . . . . . . . . . . . . . . . . . . . . . . 5 6 5.1.5 Incidence of Acanthamoeba Keratitis . . . . . . . . . . . . . . . . . . . . . . . . . . 5 7 5.1.6 Pathogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 8 5.1.7 Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 9 Health Effects Support Document for Acanthamoeba iv 5.2 Granulomatous Amoebic Encephalitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 10 5.2.1 Diagnosis and Treatment of GAE . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 12 5.2.2 Incidence of GAE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 12 5.2.3 Pathogenesis and Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 13 5.3 GAE in Domestic Animals and Wildlife . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 13 5.4 Other Infections caused by Acanthamoeba . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 13 5.5 Immunocompromised Individuals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 14 5.6 Incidence to Children . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 14 5.7 Effect of Endosymbiosis on Virulence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 15 6.0 HEALTH EFFECTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1 6.1 The Organism and its Occurrence ( Exposure) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1 6.2 Epidemiological Evidence for Acanthamoeba Keratitis Transmission by Tapwater 6 1 6.3 Resistance to Drinking Water Treatment and Disinfection . . . . . . . . . . . . . . . . . . . 6 2 6.4 Dose Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 3 6.5 Risk Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 3 7.0 ASSOCIATION OF CONTACT LENSES WITH ACANTHAMOEBIC KERATITIS . . . 7 1 7.1 Types of Contact Lenses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1 7.2 Demographics of Contact Lens Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2 7.3 Risk Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 3 7.4 Contact Lens Disinfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 5 7.4.1 Studies of Lens Disinfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 5 7.4.2 Hydrogen Peroxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 6 7.4.3 Multi Purpose Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 7 8.0 DATA GAPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 1 9.0 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1 Health Effects Support Document for Acanthamoeba v LIST OF TABLES Table 2.1 Major Waterborne/ Water based Pathogenic Protozoa . . . . . . . . . . . . . . . . . . . . . 2 1 Table 3.1 Currently Identified Species of Acanthamoeba . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1 Table 3.2 Acanthamoeba Species Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2 Table 3.3 Bacterial Endosymbionts of Acanthamoeba . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 6 Table 4.1 Occurrence of Acanthamoeba . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1 Table 5.1 Comparison of Clinical and Pathological Features of Granulomatous Amoebic Encephalitis ( GAE) and Acanthamoeba Keratitis ( AK) . . . . . . . . . . . . . . . . . . . 5 1 Table 5.2 Characteristics and Symptoms of Patients with Acanthamoeba Keratitis . . . . . . 5 3 Table 5.3 Worldwide Incidence of Acanthamoeba Keratitis . . . . . . . . . . . . . . . . . . . . . . . . 5 8 Table 6.1 Human Infection Caused by Species of Acanthamoeba . . . . . . . . . . . . . . . . . . . 6 2 Table 6.2 Mechanisms involved in Acanthamoeba Keratitis . . . . . . . . . . . . . . . . . . . . . . . 6 4 Table 7.1 History of Contact Lens Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1 Table 7.2 Types of Contact Lenses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2 Table 7.3 Wearers and Types of Contact Lenses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 3 Table 7.4 Age Distribution of Contact Lens Wearers in the United States . . . . . . . . . . . . . 7 3 Table 7.5 Risk Factors Associated with Acanthamoebic Keratitis . . . . . . . . . . . . . . . . . . . 7 4 Table 7.6 Types of Contact Lenses Associated with Acanthamoebic Keratitis . . . . . . . . . . 7 4 Table 7.7 Risk Factors for Acanthamoebic Keratitis in Contact Lens Wearers . . . . . . . . . 7 5 Health Effects Support Document for Acanthamoeba vi LIST OF FIGURES Figure 3.1 Life Cycle of Acanthamoeba Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3 Figure 3.2 Acanthamoeba trophozoite. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 4 Figure 3.3 Cysts of Acanthamoeba. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 4 Figure 3.4 Significance of Endosymbiosis to Waterborne Disease Transmission . . . . . . . . 3 7 Figure 5.1 Life Cycle of Acanthamoeba spp. and Human Infection . . . . . . . . . . . . . . . . . . . 5 2 Figure 5.2 Slit lamp view showing a paracentral complete ring infiltrate of the cornea . . . . 5 5 Figure 5.3 Normal Eye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5 Figure 5.4 Granulomatous Amoebic Encephalitis ( GAE) . . . . . . . . . . . . . . . . . . . . . . . . . . 5 11 Figure 6.1 Eye Trauma and Contact Lenses as Determinants of Susceptibility to Acanthamoeba Keratitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 5 Health Effects Support Document for Acanthamoeba vii GLOSSARY OF TERMS Amphizoic amoeba Amoeba able to live both free in nature and as pathogens in a host Anterior uveitis Inflammation of the iris and ciliary body Axenic Grown in the absence of other microorganisms Cytopathogenic effects Alteration of the appearanc of animal cells in culture due to the growth of pathogenic microorganisms Confocal microscopy Microscopy using a laser scanning fluorescent microscope which gives a digital two dimensional signal that is reconstructed into a three dimensional image Cornea The clear, transparent anterior portion of the fibrous coat of the eye Endocyst The innermost cellulose containing layer of the Acanthamoeba cyst. It may be stellate, polygonal, oval, triangular, or round.

Endosymbiosis One organism living within the other in a mutually beneficial relationship Epithelium The layer of cells forming the epidermis of the skin and the surface layer of mucous and serous membranes Exocyst The wrinkled proteinaceous outer layer of the Acanthamoeba cyst Free living Replicate in the environment and do not require a host Granulomatous Subacute opportunistic infection caused by Acanthamoeba spp. amoebic encephalitis It spreads from lung or skin lesions to the central nervous system, resulting in neurologic deficits that progress to meningoencephalitis and death Hematogenous spread Spread through the blood Keratitis Inflammation of the cornea IgA The predominant antibody class present in secretions IgG The predominant antibody present in human serum Health Effects Support Document for Acanthamoeba viii Macrophage Cells found in the body having the ability to engulf or phagocytose particulate substances ( e. g. bacteria)

Meningoencephalitis Inflammation of the brain and meninges Nodular scleritis A small aggregation of cells causing inflammation of the sclera Ocular Concerning the eye or vision Phagocytosis Ingestion ( engulfment) and digestion of bacteria Ring infiltrate Insoluble complexes formed by soluble antigens and antibodies, that can be visualized as localized rings in the corneal stroma. Diagnostic of free living amebic keratitis.

Sclera A tough, white, fibrous tissue that covers the so called white of the eye, extending from the optic nerve to the cornea Scleritis Superficial and deep inflammation of the sclera Stroma Foundation supporting tissues of an organ Stromal Concerning or resembling the stroma of an organ Subacute Between acute and chronic Uvea The second vascular coat of the eye, lying immediately beneath the sclera. It consists of iris, ciliary body, and choroid. Health Effects Support Document for Acanthamoeba 1 1 1.0 EXECUTIVE SUMMARY The Safe Drinking Water Act, as amended in 1996, requires the U. S. Environmental Protection Agency ( EPA) to publish a Drinking Water Contaminant Candidate List ( CCL). During the development of the first draft list in 1996, EPA obtained input from stakeholders including an international panel of expert microbiologists and the Science Advisory Board. The expert microbiologists' panel recommended that EPA issue a public health guidance for controlling Acanthamoeba for contact lens wearers. Acanthamoeba spp. are protozoan that are common in water and soil and have been associated with inflammation of the human cornea usually in contact lens wearers and chronic encephalitis in immune deficient individuals. The organism is transmitted by contact of the eye or possibly other body surfaces with contaminated water, air or soil. There is no evidence that it is transmitted by ingestion. EPA has developed this document to review the health effects of Acanthamoeba and the significance of water in its transmission. A guidance document providing recommendations for control of Acanthamoeba will follow. The document is organized into nine chapters and it includes Acanthamoeba history and taxonomy, occurrence and health effects, risk factors associated with Acanthamoeba, exposure particularly with contact lens users and infection prevention.

Acanthamoeba spp. are protozoa which are widespread in the environment. However, only a few species are capable of causing disease in humans. Acanthamoeba are capable of causing eye infections in persons who wear contact lenses or experience eye trauma. It is also capable of causing granulomatous amoebic encephalitis in immune deficient individuals. Acanthamoeba that cause disease are also " free living" i. e. they can reproduce in the environment without infecting a host. Those capable of causing disease are referred to as amphizoic amoeba because of their ability to live both free in nature and as pathogens in a host. Acanthamoeba has two stages in its life cycle ( cyst and trophozoite). The cyst is the environmentally resistant stage and can survive in the environment for many years. Acanthamoeba feed on bacteria, fungi, other protozoa, and cyanobacteria. They are easily grown on non nutrient agar plates seeded with Escherichia coli or Klebsiella pneumoniae.

The genus Acanthamoeba consists of as many as 20 species classified in three groups based on cyst morphology. Several species of Acanthamoeba are known to cause infections in humans. They include A. astronyxis, A. castellanii, A. culbertsoni, A. divionensis, A. griffini, A. healyi, A. rhysodes, A. hatchetti, A. palestinensis and A. polyphaga. Contaminated recreational and tap water have been implicated as sources of exposure, especially for those species causing infections of the eye. No studies are available on Acanthamoeba spp. in drinking water in the United States. Acanthamoeba are abundant in the environment, and can be found in tap water, seawater ( frequently near sewage disposal sites and outfall), air, soil, dust, vegetables, and animal wastes. Residential and public pools and spas have been documented as frequent sources of the amoebae which can survive pool and spa disinfection procedures because of their resistant cyst stages. Eye wash stations have also been shown to be reservoirs for the amoebae. Health Effects Support Document for Acanthamoeba 1 2 Two types of illnesses are most commonly associated with Acanthamoeba. These are Acanthamoeba keratitis and granulomatous amoebic encephalitis ( GAE). Keratitis occurs primarily in healthy individuals who wear contact lenses or have corneal trauma and GAE occurs primarily in immune deficient individuals. Acanthamoeba keratitis is characterized by severe ocular pain, a complete or partial paracentral stromal ring infiltrate, recurrent corneal breakdown of the epithelium, and corneal lesions. While positive diagnosis of acanthamoebic keratitis can be made by in vivo confocal microscopy, diagnostic tests usually rely on demonstrating amoebae on corneal scrapings or biopsy material, in which cysts and trophozoites can be visualized with a number of different stains. More recently, molecular techniques such as polymerase chain reaction are becoming part of the diagnostic tools for Acanthamoeba.

Risk of acanthamoebic eye infection is associated with eye trauma ( physical injury to the eye) or wearing of contact lenses in conjunction with exposure to water containing Acanthamoeba such as tapwater, hot tubs, natural springs, bottled water, and non sterile waters used to store contact lenses. Reports indicate that 85% of cases are associated with individuals who wear contact lenses. The pathogenic potential of Acanthamoeba appears to be related to certain strains with an ability to adhere to the cornea and the ability of the host to produce IgA antibodies in the tears.

Contact lenses are medical devices regulated by the Food and Drug Administration ( FDA) under the Safe Medical Devices Act of 1990. The FDA provides comprehensive directions for manufacturers of contact lens care products. It has been estimated that 34 million people in the United States, and 71 million people globally wear contact lens. Every individual who wears contact lenses can be infected with Acanthamoeba spp. when proper lens care and use of proper procedures for lens care products are not adhered to. There are various types of contact lenses. They are the daily wear soft lenses, daily wear disposable soft lenses, extended wear soft lenses, extended wear disposable soft lenses, rigid gas permeable lenses, colored soft contact lenses, and the theatrical or special effects lenses. Of the 34 million people in the United States who wear contact lenses, 80% of them wear soft contact lenses, 64% are female and 36% are male. The approximate percentage of children below the age of 17 who wear soft contact lenses is 10%. As contact lens care became easier and more convenient, people of all ages from as young as 8 years old to over 60 have been issued prescriptions to wear them. Colored contact lenses, which are often worn for cosmetic purposes, have become very popular particularly within the teen population. Teenagers frequently trade, borrow, and swap lenses. This behavior in the teen population has also added to the problem of Acanthamoeba keratitis since good hygiene may not be practiced. Treatment for Acanthamoeba keratitis includes various combinations of propamidine isethionate ( Brolene), dibromopropamidine ointment, neomycin sulfate polymixin B sulfategramicidin oral itraconazole, topical miconazole, polyhexamethylene biguanide ( PHMB), and topical clotrimazole.

Options for lens disinfection include chlorohexidine, benzalkonium chloride, and hydrogen peroxide. Of these, hydrogen peroxide is the most effective chemical disinfectant against bacteria Health Effects Support Document for Acanthamoeba 1 3 and Acanthamoeba, including trophozoites and cysts. Chlorine is not considered effective. Multipurpose solutions have been produced to clean and store lenses with a single solution without the need for neutralization of the disinfectant before lens use. Multi purpose solutions provide the easiest technique for the lens wearer to clean and disinfect the lens and better compliance results have been demonstrated. Multi purpose solutions contain a detergent with a polyquaternium or polyhexamethylene biguanide ( PHMB), in a buffered solution.

Acanthamoeba keratitis is not a reportable disease in the United States so the true incidence is not known. Published work suggests an incidence of 0.58 to 0.71 cases/ 1,000,000 in the general population, and 1.65 to 2.01/ 106 among contact lens wearers. One study in the United Kingdom reported an incidence of 149/ 106 among the general population. In contrast, the incidence of all causes of microbial keratitis ( largely bacterial) is about 400/ 106 among contact lens wearers. Worldwide, the incidence of microbial keratitis has been reported to range from 1.1 to 2,000/ 106 among contact lens wearers. Difficulties in the diagnosis of Acanthamoeba keratitis probably leads to an underestimation of the true number of cases.

Molecular based investigations have established domestic tapwater as a proven source of Acanthamoeba infection in lens wearers. The organisms have been isolated from household taps and probably feed on the microbial biofilm within the distribution system. An epidemiological study in the midwestern United States suggested that an epidemic of presumed Acanthamoeba infection was associated with municipal water supplies subjected to flooding during 1993 1994. The incidence of Acanthamoeba was ten times greater ( 1.30 vs. 14.3 cases/ 106) in areas affected by flooding. The incidence was also significantly lower if the home was supplied with tapwater from a private well. Studies suggest that the risk of Acanthamoeba keratitis may be related to concentrations of the organism present in surface waters and tapwater.

Granulomatous amoebic encephalitis ( GAE) caused by Acanthamoeba is the second major infection associated with Acanthamoeba. GAE is now recognized as a disease occurring most often in people with poor immune systems or other debilitating health problems. Predisposing factors include chemotherapy, dialysis, diabetes, treatment with steroids, smoking, or acquired immunodeficiency syndrome. The symptoms of GAE during the initial stage of the disease are indistinguishable from bacterial and viral meningitis. The amoeba is believed to enter the bloodstream, probably via the nose, lungs, or breaks in the skin following injury or trauma. Successful treatment is rare. Pentamidine, propamidine, miconazole, ketoconazole, sulfadiazine, itraconazole, fluconazole, and 5 fluorcytosine may be effective in treating GAE, and efforts to find at least a partially successful treatment are in progress.

The global incidence of recorded GAE cases due to Acanthamoeba was 120 cases as of the year 2000, 84 of those occurred in the U. S. and over 50 of the GAE cases were found in AIDS patients. An estimate of Acanthamoeba keratitis cases in the U. S. stood at 500 with over 3000 cases worldwide. There is general agreement that both GAE and keratitis have significantly increased in Health Effects Support Document for Acanthamoeba 1 4 the last 10 years in the U. S. because of the increase in the use of contact lens wearers of all ages for various reasons including athletic and cosmetic reasons, and the increase in the number of immuno suppressed individuals.

Other areas of concern with Acanthamoeba spp. in drinking water supplies is their symbiotic relationship with waterborne pathogenic bacteria that are able to grow within the cytoplasm of the protozoa. This endosymbiotic relationship with Legionella, Mycobacterium, and Pseudomonas enhances bacterial survival and resistance to disinfectants in water. It also increases the virulence of both organisms, resulting in a greater probability of causing illness. Acanthamoeba may play a significant role in the transmission of these bacteria by drinking water. Control of Acanthamoeba in distribution systems may be necessary for control of Legionella and Mycobacterium.

Acanthamoeba cysts are very resistant to inactivation by water disinfectants such as chlorine, iodine, bromine, and ultraviolet light. Doses used in drinking water would not be expected to eliminate them. The cysts of some Acanthamoeba cysts, however, are large enough to be removed by filtration. Because of their widespread occurrence in the environment, contamination of household taps, where bacteria upon which they feed are common in the biofilm, their presence would not be unexpected. Concentrations in distribution systems probably depend upon the concentration of heterotrophic bacteria.

While it is clear that a relationship exists between Acanthamoeba in water and keratitis, the role of tapwater is not clearly understood. One study suggests that municipal supplies which may have become contaminated enhanced the risk of presumed Acanthamoeba keratitis. Additional information on dose needed for infection and quantitative data on occurrence in drinking water supplies would help to better understand the potential risks to contact lens wearers and the general public. The incidence of recognized Acanthamoeba keratitis is around 1 2/ 106. The highest incidence in the U. S., which may have been linked to flooding and the use of municipal water supplies, was 14/ 106. Even if all the cases of Acanthamoeba were associated with tapwater this would be less than the 1: 10,000 risk of infection per year that EPA has set as the goal for surface water supplies.

The risk of keratitis is clearly greater for contact lens wearers. If consumers follow contact lens manufacturers' instructions and lens care product instructions for storage and rinsing of lenses, keratitis would be greatly reduced. Proper contact lens care and disinfection are essential for preventing infection by Acanthamoeba.

A significant data gap is the absence of information on the occurrence of Acanthamoeba spp. in tapwater in the United States. Information on the concentration of Acanthamoeba spp., virulence, and type of water treatment would improve the risk assessment process for drinking water. Dose response data could be developed in animals to aid in prediction of the probability of infection from exposure. Health Effects Support Document for Acanthamoeba 2 1 2.0 INTRODUCTION Acanthamoeba is a protozoan genus. Protozoa are unicellular eukaryotic animals. While protozoa are widespread in the environment, only a few are capable of causing disease in humans. Several of the pathogenic protozoa are transmitted by water, including Giardia lamblia, Cryptosporidium spp., Naegleria fowleri and certain Acanthamoeba spp ( Table 2.1).

Acanthamoeba are free living amoebae which have no defined shape. They move by pseudopods, extensions of the cell membrane into which the cytoplasm moves. They normally live in soil, fresh water, brackish water, sewage, and biosolids, feeding on bacteria, and multiplying in their environmental niche as free living organisms. They are capable of causing infections of the human skin, lungs, eye and brain, and can feed on human tissue. Because of their ability to live both free in nature and as pathogens in a host, they are also called amphizoic amoeba. This is in contrast to the Giardia and Cryptosporidium which do not replicate in the environment ( Table 2.1). These waterborne pathogenic protozoa are transmitted only by ingestion and replicate only within the host.

The genus Acanthamoeba consists of as many as 20 species classified in three groups based on their morphology ( Table 3.2). Unlike Naegleria fowleri, the most important species of Naegleria that causes human disease, several species of Acanthamoeba are known to cause infections in humans. They include A. astronyxis, A. castellanii, A. culbertsoni, A. divionensis, A. healyi, A. rhysodes, A. hatchetti, A. palestinensis and A. polyphaga. Exposure to contaminated recreational and tapwater has been implicated as a source of exposure, especially for those species causing infections of the eye.

Table 2.1 Waterborne/ Water based Pathogenic Protozoa Type Genus/ species Disease/ Symptoms Amoeboid Acanthamoeba Naegleria eye infection ( keratitis), brain infection( meningo encephalitis) brain infection( meningo encephalitis) Entamoeba hystolytica amoebic diarrhea ( liver abscess)

Flagellate Giardia lamblia diarrhea Apicomplexan Toxoplasma gondii fever, loss of fetus Cryptosporidium diarrhea Cyclospora cayetanesis diarrhea Health Effects Support Document for Acanthamoeba 3 1 3.0 GENERAL INFORMATION AND PROPERTIES 3.1 History and Taxonomy Prior to the 1950' s, amoebae such as Entamoeba histolytica were classified as parasitic ( requiring a host for replication), while species of Acanthamoeba were viewed as free living ( replicate in the environment). However, Jahnes et al. ( 1957) found that an unidentified species of Acanthamoeba could cause cytopathogenic effects in monkey kidney cell cultures, and Culbertson et al.( 1958) found that it could cause meningoencephalitis in experimentally infected animals. Results of studies with laboratory animals led to the finding that these free living amoebae had caused fatal meningitis in several patients. The term " free living pathogenic amoebae", or PFLA, has been used to describe these opportunistic pathogens. They are now referred to as amphizoic amoeba ( Page, 1967).

Taxonomy of Acanthamoeba is a contentious area. Those species now known as Acanthamoeba were previously placed in the genus Hartmanella, but in 1967 they were definitely classified as a separate genus by Page ( 1967). Pussard and Pons ( 1977) later proposed a classification based mainly on cyst morphology that identified 18 species ( Table 3.1). The species were classified into three morphologic groups ( Table 3.2). Group I has large cysts with rounded outer walls ( ectocysts) that are clearly separated from the inner walls ( endocysts). The inner and outer walls are joined, forming a star shaped structure. Group II cysts are smaller, with variable endocyst shapes. Group III cysts are smaller than Group II cysts, with poorly separated walls. The major human pathogens belong to Group II, although A. culbertsoni, from Group III, is also a recognized pathogen.

Table 3.1 Cu rrently Identified S pecies of Acanthamoeba Species Species A. astronyxis A. mauritaniensis A. castellanii A. palestinensis A. comandoni A. paradivionensis A. culbertsoni A. pearcei A. divionensis A. polyphaga A. echinulata A. quina A. gigantea A. rhysodes A. griffini A. royreba A. hatchetti A. stevensoni A. healyi A. terricola A. jacobsi A. triangularis A. lenticulata A. tubiashi A. lugdunensis Health Effects Support Document for Acanthamoeba 3 2 Table 3.2 Acanthamoeba Species Classification ( Pussard and Pons, 1977)

Group I Group II Group III A. astronyxis A. castellani A. palastinensis A. comandoni A. mauritaniensis A. culbertsoni A. echinulata A. polyphaga A. lenticulata A. lugdunesis A. pustulosa A. quina A. royreba A. rhysodes A. divionensis A. paradivionensis A. griffini A. triangularis 3.2 General Characteristics Acanthamoeba has two stages in its life cycle: the trophozoite and the cyst ( Figure 3.1). Acanthamoeba trophozoites measure 15 to 45 m m and are characterized by the presence of fine, tapering, spine like projections from the surface of the body, called acanthopodia. The acanthopodia can be periodically protruded and retracted ( Figure 3.2). The trophozoites usually have one nucleus with a large, dense nucleolus. Acanthamoeba divide by conventional mitosis, in which the nucleolus and the nuclear membrane disappear during cell division. Numerous mitochondria, ribosomes, lysosomes, and vacuoles are present within the cytoplasm. The trophozoite feeds on bacteria by engulfing them ( phagocytosis). Under adverse environmental conditions a dormant cyst is formed, which is resistant to desiccation, temperature extremes and disinfectants. The cyst is slightly smaller than the trophozoite ( 15 28 m m in length) ( Figure 3.3). It has one nucleus and is double walled, with a wrinkled proteinaceous outer ectocyst and an inner cellulose containing endocyst. The inner endocyst may be stellate, polygonal, oval, triangular or round. Pores or ostioles are present at the point of contact between the ectocyst and endocyst ( Figure 3.3).

The cyst may remain viable for many years and when it is exposed to a food source, it again assumes the trophozoite form. It is not understood how the cyst recognizes a food source. It will readily excyst in the presence of both liquid nutrients and bacteria.

Acanthamoeba are carriers of intracellular bacteria, especially Legionella species, which have the ability to reproduce within the trophozoite. It has been proposed that this may be of importance in the persistence and spread of these organisms in the environment ( King et al., 1988). Health Effects Support Document for Acanthamoeba 3 3 Figure 3.1 Life C ycle of Acanthamoeba Species Health Effects Support Document for Acanthamoeba 3 4 Figure 3.2 Acanthamoeba Trophozoite ( amebic stage). Note the characteristic spinelike acanthapodia. ( Visvesvara, 1987)

Figure 3.3 Cysts of Acanthamoeba. Note the characteristic double wall with an outerwrinkled ectocyst and an inner polygonal endocyst ( Visvesvara, unpublished) Health Effects Support Document for Acanthamoeba 3 5 3.3 Methods of Identification The identification of individual species of Acanthamoeba is based on morphological observations, but recent taxonomic studies have employed isoenzyme ( de Jonckheere, 1987) or mitochondrial DNA restriction endonuclease analysis in an attempt to form a classification system. A study of mitochondrial DNA has produced comparable results. In the first study, 33 strains, of which 30 were corneal isolates, were separated into ten groups according to restriction length pattern polymorphism.

3.4 Cultivation Acanthamoeba are easily grown on non nutrient agar plates seeded with Escherichia coli or Klebsiella pneumoniae ( Kilvington et al., 1990; Visvesara et al., 1975). One of the more common methods is to smear or streak a suitable bacterial food organism such as Escherichia coli or Klebsiella pneumoniae over the agar surface, seal the plates with tape, invert them and incubate them in boxes lined with wet paper towels to maintain humidity. Acanthamoeba will migrate across the plate using bacteria as a food source. Overproliferation of bacteria is prevented by the non nutrient agar. With incubation at 32 ° C, the migration tracks of the amoebae are usually easily visible within 48 hours, but occasionally longer incubation ( up to two weeks) is needed ( Illingworth and Cook, 1998).

Formulations for several complex liquid axenic ( bacteria free) media may be found in a publication by the American Type Culture Collection ( Nerad, 1993). Since some species of amphizoic amoeba grow at mammalian body temperatures, many labs incubate replicate cultures at room temperature, 37 º C to 45 º C, or higher.

3.5 Significa nce of Endosymbiosis Acanthamoeba feeds on bacteria in the environment trapping them within its cytoplasm, a process known as phagocytosis. Phagocytosed bacteria are usually killed and digested by the amoebae, however, some species of bacteria may grow and reproduce within the cytoplasm and become symbionts. Symbiotic relationships are beneficial to both organisms. When the bacteria have adapted to the intercellular environment of the protozoan host, the event is referred to as endosymbiosis. Both the survival and virulence of both organisms may be enhanced by this relationship ( see Section 5.7). Rowbotham ( 1980) first reported the association of the amoebae Naegleria and Acanthamoeba with the symbiont Legionella pneumophila, the causative agent of Legionnaire's disease. Several species of free living amoeba have been shown to support the growth of legionellas ( Fields, 1993) and environmental growth of legionellas in the absence of protozoa has not been documented. It is thought that the protozoa are the primary means of proliferation of these bacteria under natural conditions ( Fields et al., 1989; Hay et al., 1995). This endosymbiotic relationship can modify the virulence of Legionella ( Dowling et al., 1992). It may also be involved in the observed phenomenon that L. pneumophila can be viable but nondetectable by cultivation on agar based systems ( Connor et al., 1993). Hay and Seal ( 1994b) Health Effects Support Document for Acanthamoeba 3 6 have proposed that the latter observation may have profound implications with regard to surveillance of water systems for Legionella, especially with prevention of outbreaks of nosocomial Legionnaire's disease.

Various waterborne pathogens have been shown to develop an endosymbiotic relationship. The spectrum of pathogens able to survive and multiply to various degrees within Acanthamoeba is given in Table 3.3. For all of the organisms, Acanthamoeba are potential reservoirs and vectors, due in part to their ubiquity in the environment, their resistant cyst stages, and their potential to grow in water supplies, cooling, humidification systems, and recreational waters.

Endosymbiosis has also been shown to protect Legionella against disinfection ( Kilvington and Price, 1990), and enhance the ability of both the bacteria and protozoa to cause disease ( see Section 5.7). Thus, the presence of Acanthamoeba in drinking water distribution systems may not only add to the survival of other waterborne pathogens, but this relationship may enhance their virulence ( Figure 3.4).

Table 3.3 Bacterial Endosy mbionts* of Acanthamoeba Legionella pneumophila Mycobacterium avium Burkholderia picketti Vibrio cholerae Francisella tularensis Chlamydia pneumoniae Rickettsiales Listeria monocytogenes Fritsche et al., 1999; Ly and Miller, 1990 * live within the Acanthamoeba Health Effects Support Document for Acanthamoeba 3 7 Figure 3.4 Significance of Endosymbiosis to Waterborne Disease Transmission Health Effects Support Document for Acanthamoeba 4 1 4.0 OCCURRENCE Acanthamoeba are abundant in the environment and have been isolated from tapwater, seawater, air, soil, dust, and vegetables ( Table 4.1). They feed on bacteria, fungi, other protozoa, and cyanobacteria ( blue green algae) ( Rodriguez Zaragoza, 1994). They are found in greatest numbers where other microorganisms are most numerous.

Table 4.1 Occurrence of Acanthamoeba Source Reference Water fountains Crespo et al., 1990 Tap water ( Mexico) Rivera et al., 1979 Bottled water ( Mexico) Rivera et al., 1981 Hospital tap water Rohr et al., 1998 Eyewash stations Tyndall et al. , 1987 Freshwater ponds John and Howard, 1995 Thermal water DeJonckheere, 1979, Dive et al., 1982 Well water Jones et al., 1975 Physiotherapy tubs Penas Ares et al., 1994 Aquaria DeJonckheere, 1979 Municipal sewage Singh and Das, 1972 Ocean sewage dump site Sawyer et al., 1982 House dust Yamaura et al., 1993 Garden soil Singh, 1952 Sand box Yamaura et al., 1993 Garden vegetables Rude et al., 1984 Fish Taylor, 1977 Air conditioner Walker et al., 1986 Health Effects Support Document for Acanthamoeba 4 2 4.1 Water 4.1.1 Surface Waters <IP_ADDRESS> Freshwaters One of the early studies on the numbers of Acanthamoeba in a freshwater lake was published by O'Dell ( 1979). He noted a distinct seasonal variation in populations of A. polyphaga ranging from approximately 200/ gram ( g) to 1,000/ g of lake bottom mud during February through July, and 200/ g to 2,100/ g during the period of August through January. Peak counts were noted during August and September. Acanthamoeba castellanii was also observed in this study, but was recovered only on three occasions and did not exceed a population of 200/ g. Detterline & Wilhelm ( 1991) collected water samples from 59 sites in federally managed recreational waters of the U. S. and recovered temperature tolerant strains of Acanthamoeba from 16 of 31 sites that grew at 37 º C. Kyle and Noblet ( 1987) published a detailed account of amoebae present in a spillway reservoir in South Carolina. The authors studied the lake throughout the course of a year to record seasonal influences on amoeba populations, such as dissolved oxygen, attenuation, and water temperature. Information on amphizoic amoebae from this study showed that in the surface water they ranged from 5 to 10 amoebae / 50 milliliters ( ml) water in May, and peaked at 98/ 50 ml in July.

Asiri et al. ( 1990) tested sediments along a transect in the Potomac River ranging from non tidal waters above Washington, D. C. to tidal waters ( brackish) 0.8 m below a municipal sewage treatment plant. They identified seven species of acanthamoeba, most of which occurred in the tidal portion of the river near the sewage treatment plant. John and Howard ( 1995) processed 2,016 samples from ponds in Oklahoma and recovered 34 strains of pathogenic ( induced brain damage) amoebae with 35 percent identified as Acanthamoeba. They estimated that there was approximately 1 pathogen per 60 samples, and 1 pathogen per 3.4 liters of water. They found the highest percentage of pathogens during spring and fall, while Kyle and Noblet ( 1987) found summer and fall to be the peak periods.

<IP_ADDRESS> Seawater Acanthamoeba spp. have been occasionally detected in marine water and sediments. Most studies on Acanthamoeba spp. in marine sediments have been carried out in areas where sewage and other wastes have been disposed of at sea ( O'Malley et al., 1982; Sawyer et al., 1982). In another study, Sawyer et al. ( 1992) recovered several species of Acanthamoeba from sewagecontaminated inshore New York and New Jersey shellfish beds that periodically were closed to Health Effects Support Document for Acanthamoeba 4 3 shellfish harvesting. Munson ( 1993) recovered several species of Acanthamoeba from coastal waters of Bermuda, and noted a high frequency of recovery of Acanthamoeba spp. near sewage outfalls.

4.1.2 Tapwater and Bottled Water Acanthamoebae have been detected in tapwater and several studies have documented their occurrence, however, all of these studies have been done in countries other than the United States. Rivera et al. ( 1979) collected 25 one gallon water samples from faucets in private residences in Mexico. Flagellates were found in 84% of the samples, amoebae in 13% and ciliates in 1.9%. Although found infrequently, Acanthamoeba astronyxis and A. castellanii were recovered from the same samples. In another study, Hamadto et al. ( 1993) tested 50 tap water samples in Egypt and recovered unidentified species of Acanthamoeba from two of them. Michel et al. ( 1998) tested drinking water in a new hospital in Germany and found amoebae in 20 of 37 ( 54 %) samples; two of sixteen isolates of Acanthamoeba were pathogenic to mice. Rohr et al. ( 1998) collected water from 56 hot water taps in hospitals, also in Germany, and found amoebae in 29 ( 56 %) of them. The authors recovered five genera of cyst forming amoebae but none of them were species of Acanthamoeba. In England, Seal et al. ( 1992) isolated Acanthamoeba from five of six bathroom cold water taps supplied by storage tanks and one kitchen cold water tap supplied by the mains. When 41 strains of amoebae were recovered from 49 swab samples collected from moist areas in the hospital, such as walls, floor tiles, and sinks, 22 percent were species of Acanthamoeba. In a more recent study in Germany, Michel et al. ( 1998) recovered a species of Acanthamoeba from a hospital cold water tap. In a more recent study in Hong Kong, Houang et al. ( 2001) found that 8% of the homes were colonized with Acanthamoeba.

The common occurrence of Acanthamoeba in eye wash stations filled with tapwater containing free chlorine ( concentration of chlorine was not reported) has been reported in the United States ( Bowman et al., 1996). Acanthamoeba are able to grow in stagnant water in eye wash stations and regular flushing is required to control their numbers. The presence of free chlorine or other disinfectants was not reported in any of the previous studies.

Rivera et al. ( 1981) tested three popular brands of bottled mineral waters available in local stores in Mexico and identified Naegleria gruberi, Vahlkampfia vahlkampfi, and Acanthamoeba astronyxis. The author did not state how or if the water had received any processing before bottling. Health Effects Support Document for Acanthamoeba 4 4 4.1.3 Swimming Pools and Spas Residential and public pools and spas have been documented as frequent sources of amphizoic amoebae, including Acanthamoeba. When amoebae were first identified as a cause of meningitis, Lyons and Kapur ( 1977) tested water from 30 public pools in New York disinfected with either chlorine or bromine and recovered amoebae from 27 of them. The species were not identified but were referred to as belonging to the " Hartmannella Acanthamoeba" group, a term often used before the two genera were recognized as distinct taxonomic entities. Acanthamoeba has been in swimming pools or other bodies of water around the world, including Germany ( Janitschke et al., 1980), Mexico ( Rivera et al., 1983) and frozen swimming areas in Norway ( Brown and Cursons, 1977).

Thermal bathing pools ( spas) are also sources for potentially pathogenic amoebae ( Martinez, 1985). Brown et al. ( 1983) tested 9 thermal pools in New Zealand and identified temperature tolerant strains of Acanthamoeba from 20 percent of them. They set up 88 subsamples from the pools and found Acanthamoeba in 5 of them( 5.7 percent). Rivera et al. ( 1987) studied three resorts in Mexico that received water flowing from natural springs of thermal water. They recovered 12 strains of Acanthamoeba from cultures incubated at 42 º C to 45 º C. Two strains were identified as A. castellanii, one as A. lugdunensis and the others as Acanthamoeba spp. All were pathogenic to mice. The authors conducted a second study ( Rivera et al., 1991) and recovered A. culbertsoni and A. polyphaga from heated physiotherapy tubs. Penas Ares et al. ( 1994) tested heated water used to fill 12 spas in Spain. The water was classified as sulphurous, and temperature ranged from 34 º C to 64 º C. The authors recovered 13 strains of amoebae from 8 of the spas. Four of the 8 spas yielded A. polyphaga or A. lenticulata, with only A. polyphaga found to be pathogenic to mice. The amoebae may survive pool and spa disinfection procedures because of their resistant cyst stages.

4.1.4 Sewage and Biosolids Daggett ( 1982) published a description of potentially pathogenic Acanthamoeba and Naegleria in polluted waters with emphasis on health risks to divers. Singh and Das ( 1972) studied biosolid samples in Bombay, India and recovered strains of Acanthamoeba culbertsoni and A. rhysodes that were pathogenic to mice. Bose et al. ( 1990) extended studies on sewage in India to include Calcutta, where they isolated a pathogenic strain of A. castellanii and a non pathogenic strain of A. astronyxis. Health Effects Support Document for Acanthamoeba 4 5 4.2 Animal Wastes Bovee et al. ( 1961) tested intestinal contents from reptiles in Florida using the agar plate method and recovered amoebae from 35 of 157 fecal samples. Wilson et al. ( 1967) conducted a followup study in Florida and identified cyst forming genera of amoebae representing Acanthamoeba from water and the intestinal contents of snakes and lizards. Jadin et al. ( 1973) carried out an extensive study on wildlife in France and recovered Acanthamoeba from the feces of snakes, toads, frogs, ducks, gulls, and muskrats. The study showed that animals largely aquatic in habitat could be sources of Acanthamoeba in natural bodies of water. Franke and Mackiewicz ( 1982) discovered animals that transport Acanthamoeba in their feces by culturing A. polyphaga from the common shiner, Notropis cornatus, and the white sucker, Catostomies commersari, from streams in New York. Simitzis and Chastel ( 1982) reported finding species of Acanthamoeba in feces of small feral mammals in Brittany, Tunisia, and France.

4.3 Air, D ust and S oil Air is a carrier of dust, dirt, fungal spores, and other forms of particulate matter. During a dust storm in Zaire, Africa, Lawande et al. ( 1979) collected nasal swabs from 50 children ranging in age from 1 month to 10 years and recovered soil amoebae from 12 ( 24%) of them. Two of the twelve children harbored A. rhysodes. Lawande ( 1979) also exposed open culture plates to the atmosphere for periods of 30 minutes to 4 hours. Amoebae identified as A. castellanii and A. culbertsoni were recovered as early as 30 minutes after the plates were opened. The study throughout the 4 hour period yielded other species as well, including A. astronyxis, A. palestinensis, and A. rhysodes. Rivera et al. ( 1987) conducted similar studies during the rainy season in Mexico City, Mexico. They recovered A. astronyxis A. castellanii, A. culbertsoni, and A. polyphaga from air. In a second study of air in Mexico, Rivera et al. ( 1991) recovered nine species of Acanthamoeba. Air conditioners and cooling towers also contribute moisture and microbial pathogens including Acanthamoeba in the atmosphere ( Walker et al., 1986; Ma et al., 1990; el Sibae, 1993). Kingston and Warhurst ( 1969) conducted quantitative studies on the density of Acanthamoeba cysts in outdoor air. They recorded values of one cyst per m3 and one cyst of A. castellanii per 18.3 m3 of air.

4.4 Summary Acanthamoeba can be isolated from most aquatic environments, air, and soil. Their concentration in water is related to the number of bacteria upon which they feed. Little quantitative information is available on their concentration in water and their occurrence in distribution systems and tapwater has not been systematically studied in the United States. Recreational exposure may occur because of their presence in swimming pools, hot tubs and surface waters. Health Effects Support Document for Acanthamoeba 4 6 They may occur seasonally in greater numbers in the early spring and early fall. The occurrence of Acanthamoeba in the environment is summarized in Table 4.1. Health Effects Support Document for Acanthamoeba 5 1 5.0 HEALTH EFFECTS Two types of illnesses are most commonly associated with Acanthamoeba spp. These are Acanthamoeba keratitis ( an infection of the eye) and granulomatous amoebic encephalitis ( GAE). GAE infection is usually considered opportunistic. Keratitis occurs primarily in healthy individuals who wear contact lenses and GAE occurs primarily in immuno deficient individuals. A comparison of the clinical and pathological features of the two diseases is listed in Table 5.1.

Risk of acanthamoebic eye infection is associated with eye trauma ( physical injury to the eye) or wearing of contact lens in conjunction with exposure to water containing Acanthamoeba such as tapwater, hot tubs, natural springs, bottled water, and non sterile waters used to store contact lenses. Reports indicate that 85% of cases are associated with individuals who wear contact lenses.

Table 5.1 Comparison of C linical and Pathological Features of Granulomatous Amoebic Encephalitis ( GAE) and Acanthamoeba Keratitis ( AK)

Features GAE AK Predisposing Factors Immunodeficiency; AIDS; Debilitating chronic disease Good health, corneal trauma, contaminated contact lens wearing Epidemiology Worldwide Worldwide Usual Portals of Entry Lungs; skin; nose; neuroepithelium Corneal abrasion Incubation Period Probably weeks to months Probably days Clinical Course Prognosis Subacute or chronic ( several weeks to months); Almost always fatal Subacute or chronic Good if properly treated Clinical Symptoms and Signs Personality changes; confusion; seizures; nausea; headache; dizziness Eye pain; typical corneal ring " infiltrate"; photophobia; blurred vision Treatment Itraconazole; Miconazole; Sulfametazine; Pentamididine IV ( in vitro) Polyhexamethylene biguamide; Propamidine isethionate Health Effects Support Document for Acanthamoeba 5 2 Figure 5.1 Life cycle of Acanthamoeba spp. and Human Infection Health Effects Support Document for Acanthamoeba 5 3 Granulomatous amebic encephalitis or GAE is a chronic illness of the central nervous system that affects the brain and is associated with Acanthamoeba spp. It is an infection primarily of the immunocompromised individual which usually leads to death.

5.1 Eye Infections ( Acanthamoeb ic Keratitis)

Acanthamoeba species cause acanthamoebic keratitis, a painful, vision threatening disease of the cornea. The infection is associated with minor corneal trauma or the use of contact lenses in normal, healthy people. Males and females are equally affected. Acanthamoeba keratitis is characterized by severe ocular pain, a complete or partial paracentral stromal ring infiltrate, recurrent corneal breakdown of the epithelium and a corneal lesion refractory to commonly used ophthalmic antibacterial medication. Clinical features of the disease are in Table 5.2.

Table 5.2 Characteristics and Symptom s of Patients with Acanthamoeba Keratitis CYoung, healthy individuals C Soft contact lens wearers C Non preserved or non sterile solution used for storage of contact lens C Eye trauma C Usually one eye affected C Extreme eye pain C Corneal breakdown of the epithelial C Late in the infection, a corneal ring infiltrate is seen Some species of Acanthamoeba were not found to be associated with eye disease until the early 1970' s. Jones et al. ( 1973), Jones et al. ( 1975), and Visvesvara et al. ( 1975) described the case of a rancher who scraped his eye while bailing hay and rinsed it with tap water pumped into his house from a well that used unfiltered river water. The authors also described an infection in a young female nurse who had no history of eye disease, and a fatal infection in a 7 year old boy who had played in drainage ditches near his home. Nagington et al. ( 1974) described an eye infection in a 32 year old schoolteacher who did not have a history of exposure to contaminated water, and a second fatal case in a 59 year old farmer who was hit in the eye by a tree branch. Jones et al. ( 1975) also described a case involving a 58 year old farmer who had been exposed to Health Effects Support Document for Acanthamoeba 5 4 dust while baling barley on his farm. The infection failed to respond to treatment and had to be surgically removed.

Other cases of physical damage include irritation by an insect ( Hamburg and DeJonckheere, 1980), contamination by barley dust ( Jones et al., 1975), and wind surfing ( Volker Dieben et al., 1980). The effects from eye trauma ranged from successful treatment, corneal replacement, loss of the affected eye and, rarely, death of the patient. Jones et al. ( 1975) described a fatal case in a young boy who was suspected of playing in a watering trough for cattle.

The number of eye infections reported in the 1970' s generally were unique case histories involving injury. All of this changed when some of the eye infections thought to be of viral origin were found to be caused by Acanthamoeba ( MMWR, 1987). Ormerod and Smith ( 1986) reviewed the histories of 42 cases of keratitis in California that occurred between 1977 and 1984 and suggested that it was likely that extended wear lenses might increase the risk of microbial keratitis. Stehr Greene et al. ( 1987) conducted a case control study to obtain information on the role of contact lens sanitary practices on injury to the eye. They studied 27 patients with keratitis and 81 uninfected individuals ( controls) in order to compare lens care practices. Patients with keratitis were found more likely to use homemade solutions than controls ( 78 versus 17 percent) and were more likely to wear lenses while swimming ( 63 versus 30 percent). The authors found that microbial contaminants other than Acanthamoeba were present in 1 of 59 commercial saline solutions, 11 of 11 homemade solutions, and 23 of 29 bottles of non sterile distilled water. Thus, there is little doubt that microorganisms in non sterile cleansing solutions may become established in contact lens cases, perhaps on the lenses themselves, and lead to serious eye disease. Badendoch ( 1991), Martinez and Visvesvara ( 1997) have reviewed most of the literature on amoebic eye diseases beginning with some of the earliest recognized cases and noted that successful outcomes depended on early diagnosis and treatment. Martinez and Visvesvara ( 1997) estimated that, as of January 1996, more than 750 cases of amoebic keratitis have been reported worldwide.

There are several important risk factors associated with acanthamoebic keratitis. The vast majority of patients have at least one of these identifiable factors, which include corneal trauma, exposure to contaminated water, and contact lens use. Approximately 71 to 85% of patients with acanthamoebic keratitis are contact lens wearers ( Moore and McCulley, 1989; Moore et al., 1985).

No single type of contact lens has been excluded from association with acanthamoebic keratitis. People with daily wear soft contact lenses account for approximately 75% of the cases, people with extended wear contact lenses account for about 14%, people with hard contact lenses account for about 6%, and people with rigid gas permeable lenses account for about 4% ( Moore et al., 1985). In another study, Stehr Green et al.( 1987) reported that most patients ( 95%) had at least one risk factor for acanthamoebic keratitis, the 85% who wore contact lenses, most wore Health Effects Support Document for Acanthamoeba 5 5 daily wear ( 56%) or extended wear soft ( 19%). Some patients ( including both contact lens wearers) ( 26%) had a history of corneal trauma before developing acanthamoebic keratitis, and 25% of patients had a history of exposure to contaminated water.

Two studies have identified tapwater washing of lens cases in cases of Acanthamoeba ( Seal et al., 1997, Ledee et al., 1996). Ledee et al., 1996 using molecular fingerprinting techniques established domestic tapwater in the United Kingdom as the source of contamination in contact lens wearers. Similarly, contact lens wearers who have been exposed frequently to hot tubs or natural springs are at risk of developing acanthamoebic keratitis ( Wilhelmus and Jones, 1991).

5.1.1 Symptoms of Acanthamoeba Keratitis Clinical symptoms are usually a history of pain and the formation of a whitish halo or ring infiltrate around the periphery of the cornea ( Figure 5.2). Although most cases present a history of contact lens wear, the infections are also associated with a foreign object or physical trauma in the affected eye. A normal eye is shown in Figure 5.3. Health Effects Support Document for Acanthamoeba 5 6 5.1.2 Diagnosis of Acanthamoeba Keratitis While positive diagnosis of acanthamoebic keratitis can be made by in vivo confocal microscopy, diagnostic tests usually rely on demonstrating amoebae on corneal scrapings or biopsy material ( Seal et al., 1996). Samples of corneal epithelium and any infiltrated stroma are removed under local anesthetic, and contact lenses and storage cases may also be cultured. The most common method is to inoculate the sample into the center of a non nutrient agar plate seeded with E. coli ( Singh and Petri, 2000). With incubation at 32 ° C in air, migration tracks are usually visible within 48 hours. Positive identification requires some experience, and it is useful to incubate a control plate that is not inoculated with a clinical specimen.

5.1.3 Identification Procedures Standard methods for morphological characterization, isoenzyme electrophoresis, immunological techniques, and temperature tolerance tests have been published and widely used ( Singh and Petri, 2000). Results obtained by using one or more of these techniques, coupled with animal pathogenicity tests, and the shape and size of cysts, are often adequate for identifying more commonly occurring species of Acanthamoeba.

Corneal biopsy of infected eye are usually sufficient for confirming infection by amphizoic amoebae. However, it may be possible to make an identification of genus when distinctive double walled wrinkled cysts suggest a Group III species of Acanthamoeba. When amoebae from corresponding pieces of tissue appear on culture plates, the cysts are often distinctive enough to place the organism in Acanthamoeba. Keys to soil amoebae ( Page, 1976; 1988) or photographs ( Pussard and Pons, 1977), often are sufficient for identifying some of the wellknown species. Biochemical methods for obtaining isoenzyme profiles ( deJonckheere and Michel, 1988) are extremely useful in combination with morphological features for identifying most amoebae ( Sawyer, 1992). Griffin ( 1972) used thermotolerance as one method for screening amoebae for pathogenicity. Pathogenicity can be assessed by a number of methods ( see Section 5.1.6).

5.1.4 Treatment of Acanthamoebic Keratitis In the first 10 years after the emergence of acanthamoebic keratitis as a clinical problem, treatment was usually unsatisfactory, employing a wide variety of topical agents in combination. In 1985, Wright et al. reported successful medical treatment using propamidine isethionate ( Brolene) 0.1%, an aromatic diamidine, applied topically with dibromopropamidine ointment 0.15%, and followed by treatment with neomycin when signs of toxicity occurred. The success of the treatment was attributed to the amoebicidal activity of both propamidine and dibromopropamidine, although subsequently dibromopropamidine was generally omitted from the regimen. Further experience showed that a medical cure with propamidine therapy was most Health Effects Support Document for Acanthamoeba 5 7 likely to be achieved if treatment began early in the course of the disease ( Moore and McCulley, 1989). Propamidine was generally combined with neomycin, initially instilled hourly and tapered slowly over several months after improvement was noted. However, in some patients results were still poor, and more effective compounds were sought ( Ficker, 1988). Successful treatment using propamidine with miconazole 1% ( often with neomycin sulfate polymixin B sulfate gramicidin) has been reported ( Berger et al., 1990), as has combination therapy with oral itraconazole, with topical miconazole 0.1% and debridement ( Ishibashi et al., 1990). Another combination regimen is topical clotrimazole 1 2% with propamidine and neomycin sulfatepolymixin B sulfate gramicidin; in a series reported recently a medical cure was achieved in 11 of 14 patients with eye infections using this combination ( D'Aversa et al., 1995).

In the early 1990' s, in vitro sensitivity studies showed that the cationic disinfectant polyhexamethylene biguanide ( PHMB) was highly effective in killing both cysts and trophozoites, and in 1992 Larkin et al. reported its successful clinical use at a concentration of 0.02%. The main theoretical advantage of PHMB over other compounds seems to be its consistently high cysticidal activity against a number of strains, compared with other compounds that may be active against some strains but relatively ineffective against others. Another factor is that in contrast to propamidine, PHMB does not appear to be associated with toxicity problems ( Johns et al., 1988). Clinical experience with PHMB ( usually in combination with propamidine) has shown that if used early enough in the course of the disease the prognosis is very good, and penetrating keratoplasty is unlikely to be necessary ( Illingworth et al., 1995).

Recently the use of the diamidine derivative hexamidine, which appears to have a greater cysticidal activity than propamidine, has been reported ( Brasseur et al., 1994). The use of chlorohexidine 0.02% as an alternative to PHMB has also been reported, resulting in a medical cure in 11 of 12 patients ( Seal et al., 1996).

5.1.5 Incidence of Acanthamoeba Keratitis Acanthamoeba keratitis is not a reportable disease in the United States so the true incidence is not known. Published work suggests an incidence of 0.58 to 0.71 cases/ 1,000,000 in the general population, and 1.65 to 2.01/ 106 among contact lens wearers ( Schaumberg et al., 1998). One study in the United Kingdom reported an incidence of 149/ 106 among contact lens wearers ( Seal, 2000). A summary of studies reporting the incidence of Acanthamoeba keratitis is shown in Table 5.3. The incidence of all causes of microbial keratitis ( largely bacterial) is about 400/ 106 among contact lens wearers. Worldwide, the incidence of microbial keratitis has been reported to range from 1.1 to 2,000/ 106 among contact lens wearers ( Cheng et al., 1999). Difficulties in the diagnosis of Acanthamoeba keratitis probably lead to an underestimation of the true number of cases. An estimate of Acanthamoeba keratitis known cases in the U. S. stood at 500 with over 3000 cases worldwide ( Martinez and Visvesvara, 2001). Health Effects Support Document for Acanthamoeba 5 8 Table 5.3 W orldwide In cidence of Acanthamoeba Keratitis Incidence per 1,000,000 Population Country Year( s) Reference 1.65 to 2.01 Contact Lens Wearer ( CLW) USA 1985 1987 Schaumberg et al., 1998 1.1 CLW Netherlands 1996 Cheng et al., 1999 149 CLW UK 1996 Seal, 2000 0.58 to 0.71 General Population ( GP) USA 1985 1987 Schaumberg et al., 1998 1.40 GP UK 1996 Radford et al., 1998 1.30 GP  Iowa well water USA 1993 1994 Meier et al., 1998 14.3 GP  during flooding municipal systems USA 1993 1994 Meier et al., 1998 5.1.6 Pathogenicity The pathogenesis of acanthamoebic keratitis has been suggested to follow two pathways ( Alizadeh et al., 1995). The first pathway is restricted to the epithelium without involvement of the stoma and has a good prognosis. The second pathway culminates in the parasites entering the stoma, resulting in extensive necrosis, and edema. The first step in the initiation of infection is the attachment to the epithelial surface. Amoebae bind to the corneal surface and produce epithelial thinning and necrosis.

The pathogenicity of Acanthamoeba spp. is related to its ability to attach to corneal epithelial cells. Khan ( 2001) found that Acanthamoeba exhibited higher number of acantodia ( structures associated with the binding of amoeba to the target cells in the eye) as compared to nonpathogenic Acanthamoeba. Additional results indicated that phagocytosis occurs in the pathogenic amoeba by formation of amoebastone ( characteristic of amoeba phagocyte) and that Acanthamoeba phageocytosis may be both an efficient means of obtaining nutrients and a significant factor in pathogenesis of Acanthamoeba infections. Khan et al. ( 2001) differentiated pathogenic Acanthamoeba by their ability to produce cytopathogenic effects ( CPE) on corneal Health Effects Support Document for Acanthamoeba 5 9 epithelial cells in culture. They also reported that pathogenic Acanthamoeba showed growth on higher osmolarity ( one molar mannitol) while growth of non pathogens was inhibited. The pathogenic potential of A. castellani isolates was correlated with the ability to bind to the corneal epithelium, respond chemotactically to corneal endothelial extracts, elaborate plasminogen activators, and produce cytopathogenic extracts ( van Klink et al., 1992).

The 18S rRNA gene ( Rns) phylogeny of Acanthamoeba has been investigated as a basis for improvements in the nomenclature and taxonomy of the genus ( Stothard et al., 1998). Twelve linages referred to as T1 T12 have been identified with most of the keratitis causing strains belonging to group T4 ( Stothard et al., 1998; Walochink et al., 2000). More recently type T6 has also been reported to be associated with keratitis ( Walochik et al., 2000).

Another factor in the pathogenicity of Acanthamoeba may be an individuals ability to produce antibodies in tears ( Alizadeh et al., 2001). The presence of serum antibody in 50 to 100% of the population suggest that exposure to Acanthamoeba species is ubiquitous ( Cursons et al., 1980; Cerva, 1989). However, patients with Acanthamoeba keratitis have significantly higher anti Acanthamoeba IgG antibody titers than heathy subjects ( Alizadeh et al., 2001). In contrast anti Acanthamoeba tear IgA was significantly lower in patients with Acanthamoeba keratitis in comparison with healthy subjects. This suggests that a low level of anti Acanthamoeba IgA antibody in the tears appears to be associated with Acanthamoeba keratitis.

In summary, the pathogenic potential of Acanthamoeba appears to be related to certain strains and the ability of the host to produce IgA antibodies in the tears.

5.1.7 Immunity The presence of serum antibody in 50 to 100% of the population suggests that exposure to Acanthamoeba species is common. ( Cursons et al., 1980; Cerva, 1989). These antibodies were shown to be capable of neutralizing cytopathogenic effects of Acanthamoeba ( Ferrante, 1991). Patients with Acanthamoeba keratitis have a significantly higher anti Acanthamoeba IgG antibody titer than healthy subjects ( Alizadeh et al., 2001). In contrast anti Acanthamoeba tear IgA was significantly lower in patients with Acanthamoeba keratitis in comparison with healthy subjects. This suggests that a low level of anti Acanthamoeba IgA antibody in the tears appears to be associated with Acanthamoeba keratitis. Persist corneal and scleral inflammation observed following cases of Acanthamoeba keratitis is not always caused by active amoebic infection but can be due to persisting acanthamoebic antigens. Yang et al. ( 2001) found that Acanthamoeba cysts were found to persist for up to 31 months in the eye after treatment although trophozoites were no longer present. They hypothesized that Acanthamoeba cysts can remain in corneal tissue for extended periods of time and may cause persistent inflammation in the absence of active amoebic infection. Health Effects Support Document for Acanthamoeba 5 10 The feasibility of inducing protective immunity to Acanthamoeba keratitis has been tested in a pig model ( Alizadeh et al., 1995). It was shown possible to induce immunity in 50% of the animals by subconjunctival injection of the parasites, and in 100% by a combination of intramuscular and subconjunctival injection, whereas corneal infection alone did not confer immunity to subsequent infection.

5.2 Granulomatous Amoebic En cephalitis Granulomatous amoebic encephalitis ( GAE) caused by Acanthamoeba spp. is the second major infection associated with Acanthamoeba. GAE is a chronic, progressive disease of the central nervous system occurring most often in persons with poor immune systems or other debilitating health problems. Predisposing factors include chemotherapy, dialysis, diabetes mellitus, treatment with steroids, chronic alcoholism, smoking, bone marrow or renal transplantation, or acquired immunodeficiency syndrome ( Marciano Cabral et al., 2000). Chronic skin infections have been reported from patients with GAE. However, it is not known whether skin lesions provide the primary site of infection or represent terminal dissemination of Acanthamoeba from the lungs to other sites ( Marciano Cabral et al., 2000). In the majority of AIDS patients, skin lesions and sinusitis are common features. It may be caused by A. astronyxis, A. palestinensis, A. culbertsoni and A. castellanii. It spreads from lung or skin lesions to the central nervous system, resulting in neurologic deficits that progress over days or weeks to meningoencephalitis and death.

Another free living amoeba, Naegleria fowleri, was later discovered to cause an aseptic meningitis that was usually fatal ( Ma et al., 1990). The term primary amoebic meningoencephalitis, or PAM, was proposed for infection by Naegleria ( Butt, 1966), and the term granulomatous amoebic encephalitis, or GAE, was proposed for infections by Acanthamoeba ( Martinez, 1980). The two disease entities differ since PAM occurs most often in young people, is associated with swimming and has a rapid onset of symptoms. In contrast, GAE occurs most often in patients with poor immune systems or patients suffering from long standing health problems regardless of age. Granulomatous amoebic encephalitis caused by Acanthamoeba or Balamuthia is now recognized as a disease occurring most often in persons with poor immune systems or suffering from some other debilitating health problem ( e. g., alcoholism, diabetes, smoking or acquired immunodeficiency syndrome [ AIDS]) ( Figure 5.4). The amoebae are believed to enter the bloodstream, probably via the nose, lungs, or breaks in the skin following injury or trauma. They then affect various organs by hematogenous spread. Health Effects Support Document for Acanthamoeba 5 11 Figure 5.4 Granulomatous amoebic encephalitis ( GAE). Section through the brain of a fatal case caused by Balamuthia mandrillaris ( Photograph courtesy of Dr. Julio Martinez, University of Pittsburgh).

Balamuthia has been identified in approximately 40 patients in the United States ( U. S.), including > 10 with AIDS infection ( Martinez et al., 1997, Visvaresvara, 2001). In contrast, Acanthamoeba has accounted for approximately 84 (~ 50 with AIDS) cases in the U. S. and 120 worldwide ( Martinez et al., 1997, Visvaresvara, 2001). The disease may be the end result of long term injury. Fatal infections probably occur in individuals with extensive damage to the central nervous system and internal organs prior to the manifestation of overt clinical symptoms.

The exact pathway of amoebae entering the brain is difficult to determine since, in most cases with a fatal outcome, there has been a history of predisposing factors. It is believed that the amoebae are spread throughout the body via blood vessels ( hematogenous spread), after entry through the nasal passages, lower respiratory system or breaks in the skin caused by injury ( Ma et al., 1990). Patients who have been treated for GAE range from children to elderly adults with a clinical history of illness ranging from about 1 week to 6 months ( Martinez et al., 1977). Symptoms of neurological disease upon admission to a hospital are varied, including headache, drowsiness, low grade fever and stiffness of the neck. Other symptoms that may appear early in the disease are personality changes, seizures, nausea, vomiting or lethargy ( Martinez and Visvesvara, 1991). Thorough diagnostic procedures are necessary to recognize amoebic meningoencephalitis because upon initial examination, the disease is not always easy to Health Effects Support Document for Acanthamoeba 5 12 distinguish from bacterial meningitis, tuberculous meningitis, brain tumors or viral meningitis ( Martinez and Visvesvara, 1997). Martinez and Janitschke ( 1985) reviewed 33 cases of GAE and listed several illnesses associated with the patients who had the disease. They included skin ulcers, cirrhosis of the liver, hepatitis, pneumonitis, renal failure, collagen connective tissue disease and pharyngitis. Predisposing factors mentioned by the authors included chemotherapy, radiation treatment, steroids, broad spectrum antibiotics, alcoholism, splenectomy and peritoneal dialysis.

5.2.1 Diagnosis and Treatment of GAE Patients with confirmed GAE usually are chronically ill, immunosuppressed, or debilitated by other causes. By the time a diagnosis has been made, the central nervous system may have been invaded, probably via the nasal passages, respiratory tract or skin ( Martinez, 1993). The diagnosis may be questionable at first because of the possibility of brain tumor, abscess or intracerebral hematoma ( Visvesvara et al., 1997). Successful treatment is rare and infection usually results in the death of the patient. In vitro studies have shown that diamidine derivatives such as pentamidine, propamidine, miconazole, ketoconazole and 5 fluorocytosine may be effective in treating GAE ( Martinez et al., 1997). There are some occasions when skin nodules harboring Acanthamoeba are detected prior to spreading to internal organs and the central nervous system. Visvesvara et al. ( 1997) suggested that when skin nodules or ulcers are present, treatment may be tried using topical chlorhexidine gluconate and intravenous pentamidine.

In spite of the poor prognosis for most patients with GAE, efforts to find at least a partially successful treatment are in progress. A new class of peptide compounds called magainins that may have amoebostatic and amoebicidal properties when used with other amoebicidal agents ( Martinez et al., 1997, Schuster and Jacob, 1992). Schuster and Visvesvara ( 1998) tested antimicrobials and phenothiazine compounds against amphizoic amoebae and found the levels affecting them probably were too high for clinical use. In other efforts, Chu et al. ( 1998) studied the effects of plant extracts that were amoebicidal or induced encystment.

5.2.2 Incidence of GAE The global incidence as of 2000 stood at 120 cases of recorded GAE cases, 84 of those occurred in the U. S. and over 50 of the GAE cases were found in AIDS patients ( Martinez and Visvesvara, 2000). There is general agreement that both GAE and keratitis have increased in the last 10 years in the U. S. because of the increase in the use of contact lens wearers of all ages for various reasons including athletic and cosmetic, and the increase in the number of immunosuppressed individuals ( Marciano Cabral et al., 2000; EPA, 1998). Health Effects Support Document for Acanthamoeba 5 13 5.2.3 Pathogenesis and Immunity The pathogenesis of GAE is complex and poorly understood ( Martinez and Visvesvara, 1997). In GAE, the immunity is predominantly T cell mediated, therefore the dimunition of CD+ and T helper lymphocytes, as occurs in AIDS patients, enables the proliferation of free living amebas. Ulceration of the skin containing both amebic trophozoites and cysts suggests also the portal of entry into the bloodstream. In experimental animals, the olfactory neuroepithelium has also been found to be a possible portal of entry ( Janitschke et al., 1996). The incubation period of GAE is unknown but is probably longer than 10 days. The ability of the Acanthamoeba to produce necrosis of the brain tissue is probably due to an enzymatic action induced by lysosomal hydrolases and phospholipase that can degrade phopholipids of the myelin sheaths ( Martinez and Visvesvara, 1997).

Studies in mice have demonstrated that it is possible to immunize animals against Acanthamoeba meningoencephalitis ( Culberton, 1971; Rowan Kelly and Ferrante, 1984). Animals immunized intraperitoneally with sonicated trophozoites of A. culbertsoni were highly resistant to intranasal infection with the organism. Those immunized with a non pathogenic A. culbertsoni or A. polyphaga were not protected against infection with A. culbertsoni.

5.3 GAE in Domestic A nimals and W ildlife Several reports of amphizoic amoebae in animals appeared in the literature at about the same time as they were found in fatal infections in humans. The principal difference between human and animal infection is that infection in humans occurs primarily in persons with deficient immune systems or those taking immunosuppressive drugs, this is not found in cases involving animals. Kadlec ( 1978) carried out one of the most extensive surveys of infection in domestic animals by amphizoic amoeba. He identified Acanthamoeba spp. from bulls, cows, a rabbit, pigeons and turkeys. Infections in animals probably occur by the same routes as reported for humans. It has also been described in dogs by several investigators ( Ayers et al., 1972, Bauer et al., 1993). Infections in the lung of water buffalo and bulls could have been nasopharyngeal from drinking unclean water ( Dwivedi and Singh, 1965, McConnell et al., 1968).

Evidence for water as a source of infection in animals by Acanthamoeba is found in reports of the amoebae in the gills, spleen, urinary bladder or blood of wild caught and ornamental fish ( Taylor, 1977, Dykova et al., 1996, Booton et al., 1999).

5.4 Other Infections Caused by Acanthamoeba Occasional infections by Acanthamoeba spp. have included a purulent discharge from an ear ( Lengy et al., 1971), a granulomatous skin lesion ( Gullet et al., 1979), rhinosinusitis in an AIDS Health Effects Support Document for Acanthamoeba 5 14 patient ( Teknos et al., 2000) and possible association with intestinal disorders ( Hoffler and Rubel, 1974; Mehta and Guirges, 1979; Thamprasert et al., 1993).

5.5 Immunocompromised Individ uals Several reports of Acanthamoeba infection in AIDS patients involved the skin, as well as other tissues and, in most cases, there was a fatal outcome in spite of treatment. In AIDS patients it is not always absolutely clear whether the AIDS virus or the amoebae were the primary cause of death. The infection with free living amoebas is a terminal event. Individuals with deficient immune systems, whether natural or acquired, represent a segment of the population that are most likely to succumb to infections with microbial pathogens including amphizoic amoebae. Gonzalez ( 1986) reported a case resulting in death in a 29 year old patient with AIDS. At autopsy, amoebae were found in the paranasal sinuses, a calf nodule, and in an abscess of the left leg, but not in the brain. The following year Wiley et al. ( 1987) examined a 34 year old patient with a history of nasopharyngeal allergies and infections with Giardia lamblia and Cryptosporidium spp. The patient underwent an appendectomy and developed a hard skin nodule above the surgical scar. The patient stated that he had noticed painful skin lesions prior to surgery. At autopsy, amoebae were found in the brain and the skin. Tissue fragments placed in kidney cell tissue cultures yielded amoebae identified as Acanthamoeba culbertsoni. Another case involving skin infection was reported by Friedland et al. ( 1992). They treated an AIDS infected 8 year old Hispanic male who died of the infection. The patient had a persistent nasal discharge and skin nodules that eventually became ulcerated and 2 to 4 mm deep prior to death. Gordon et al. ( 1992) described a fatal case in an AIDS patient caused by A. polyphaga, and Gardner et al. ( 1991) described a case probably caused by A. rhysodes. Other fatal cases in AIDS patients followed in 1994 ( Park et al.), and 1996 ( Telang et al., 1996).

Visvesvara et al. ( 1983) described a fatal case of GAE that involved a patient with a liver transplant. Twenty six days after the transplant, the patient was readmitted to the hospital with pneumonia and cytomegalovirus infection. At autopsy, amoebae were noted in the brain, lungs, blood vessel walls, adrenal and thyroid glands, lymph nodes, skin and breast tissue. Borochovitz et al. ( 1981) identified A. castellanii from a bone graft in a diseased mandible. Anderlini et al. ( 1994) described two cases of fatal amoebic encephalitis in patients with leukemia, who had received bone marrow transplants.

5.6 Incidence to Children Children do not appear more likely to develop ocular Acanthamoeba infections. Only 13% of all contact lens wearers are under 17 years of age, but the potential for keratitis may be increasing in children because of color lens swapping by teenagers ( Contact Lens Council, 2000) ( Figure 5.5). In general all types of microbial keratitis occur less in childhood and are largely associated with trauma or preexisting corneal disease ( Cruz et al., 1993). Health Effects Support Document for Acanthamoeba 5 15 5.7 Effect of Endosymbiosis on Virulence Acanthamoeba spp. has been demonstrated to develop endosymbiotic relationships with a number of waterborne bacteria, including Legionella pneumophila and Mycobacterium avium ( Table 3.3). This relationship may be important both in the growth and survival of these opportunistic pathogens in drinking water systems, and in their ability to cause disease in humans.

Cirillo et al. ( 1997) found that Mycobacterium avium replicates within Acanthamoeba castellanii and that this association enhanced both the entry and intracellular replication compared to the growth of the bacteria in broth culture. Furthermore, amoeba grown M. avium was also more virulent in a mouse model. They also found that the highest growth rate of the M. avium in the amoebae was near 37 ° C. From this observation, they suggested that if growth of M. avium in water environments occurs primarily within protozoa, the fact that M. avium has temperaturedependant growth in amoebae may explain why M. avium infections are more frequently associated with warm water supplies. It was also found that non pathogenic strains of Mycobacterium were readily killed within the amoeba.

Cirillo et al., 1999 found Legionella pneumophila grown in A. castellanii to be at least 100 fold more invasive for macrophages than when grown on agar. They also provided evidence that amoeba grown L. pneumophila expressed different proteins that may have been related to its enhanced invasiveness. The authors also suggested the replication of L. pneumophila in protozoans present in domestic water supplies may be necessary to produce bacteria that are competent to enter mammalian cells and produce human disease. A recent study has suggested that endosymbiosis enhances the virulence of the Acanthamoeba. Fritsche et al. ( 1998) reported that endosymbiont infected amoebae produced a statistically significant enhancement in cellular destruction of human embryonic tonsilar ( HET) cell monolayers in comparison to uninfected amoeba. Neither the bacteria or Acanthamoeba alone were capable of producing cellular destruction ( i. e. cytopathic effects). Whether such enhanced pathogenic effects occurs in clinical Acanthamoeba infections is unknown. Health Effects Support Document for Acanthamoeba 6 1 6.0 HEALTH EFFECTS 6.1 The Organism and its Occurrence ( Exposure)

Certain species of the genus Acanthamoeba have been associated with eye disease in humans. Five species demonstrated to be associated with eye disease are listed in Table 6.1. The majority of the infections ( 85%) in the United States are associated with the use of contact lenses, and the remainder with some trauma to the eye ( Stehr Green et al., 1987). Infection results from the exposure to Acanthamoeba through improper storage of lenses, wetting of the lenses with unsterile solutions, improper disinfection of lenses, or swimming while wearing contact lenses. One epidemiological study suggests that increased risk may exist from municipal supplies which have been subjected to flooding ( Meier et al., 1998). The concentration of free living amoebae in surface waters may vary seasonally creating a greater exposure at certain times of the year. Acanthamoeba is common in the aquatic environment ( see section 4.0) and its cyst form is resistant to inactivation by chlorine ( Radford et al., 1998). Wetting or storage of lenses in tapwater appear to be the most significant route of exposure for contact lens wearers.

6.2 Epidemiological Evidence for Acanthamoeba Keratitis Transmission by Tapwater Molecular based investigations have established domestic tapwater in the United Kingdom as a proven source of Acanthamoeba infection in lens wearers ( Ledee et al., 1996). The organisms have been isolated from household taps and probably feed on the microbial biofilm within the distribution system. An epidemiological study in the midwest United States suggested that an epidemic of presumed Acanthamoeba infections was associated with municipal water supplies subjected to flooding during 1993 1994 ( Mathers et al., 1996; Meier et al., 1998). The incidence of presumed Acanthamoeba was ten times greater ( 1.30 vs. 14.3 cases/ 106) in areas affected by flooding. The incidence was also significantly lower if the home was supplied with tapwater from a private well. In both of these studies the authors used tandem scanning confocal microscopy and confirmatory cytopathologic findings to diagnose the cases. However, the authors were unable to culture Acanthamoeba from individuals with keratitis. The authors suggested several reasons for their failure to culture the organism including ( 1) the infections were caused by a new species with different growth requirements ( 2) the inoculum was insufficient ( 3) an inhibitor was present ( 4) the organisms were present but non viable and ( 5) the infections were caused by another organism. Health Effects Support Document for Acanthamoeba 6 2 Table 6.1 Human Infection Caused by Species of Acanthamoeba Species of Acanthamoeba CNS infection Eye infection Other tissues Reference A. astronyxis X Adrenal, lymph node, sinus, skin, thyroid Gullett et al. ( 1979)

A. castellanii X X Lung, prostate, bone, muscle, sinus, skin Martinez ( 1982) Martinez et al. ( 1977) Moore et al. ( 1985) Borochovitz et al. ( 1981) Gonzalez et al. ( 1986)

A. culbersoni X X Liver, spleen, uterus, skin Martinez et al. ( 1977) Wiley et al. ( 1987) Mannis et al. ( 1986) May et al. ( 1992)

A. divionensis X DiGregorio ( 1992)

A. griffini X Ledee et al. ( 1996)

A. hatchetti X Cohen et al. ( 1985)

A. healyi X Kim et al. ( 2000)

A. palestinensis X Ofori Kwakye et al. ( 1986)

A. polyphaga X Singh and Petri ( 2000)

A. rhysodes X X Singh and Petri ( 2000) CNS  Central Nervous System 6.3 Resistance to Drinking Water Treatment and Disinfection No studies could be found on the effectiveness of drinking water treatment on the removal of Acanthamoeba cysts or trophozoites. Given the large size of the trophozoites ( 15 to 45 µ m) and cysts ( 15 to 28 µ m) they would be easily removed by filtration in a conventional water treatment plant. Their isolation from tapwater suggests that they can certainly colonize taps and feed on bacteria in the biofilm in distribution systems. De Jonckheere and Van de Voorde ( 1976) reported Acanthamoeba cysts to be very resistant to inactivation by chlorine, bromine, and Health Effects Support Document for Acanthamoeba 6 3 iodine. The chlorine resistance of two different strains varied considerably. A 99.99% ( 4 log10) inactivation of a more sensitive strain was achieved with 16mg/ liter within one hour. A 4 log10 decrease was not achieved after 24 hours with 6 mg/ liter.

The cysts have also been found to be very resistant to ultraviolet light. Change et al. ( 1985) found the cysts of A. castellanii to be more resistant than Bacillus subtilis spores. A dose of approximately 70 mW sec/ cm2 was required for a 99% ( 2 log10) inactivation of the cysts. The viability of the cysts was detected with a plaque assay on a lawn of Escherichia coli bacteria, requiring both excystation and growth of the organism.

In contrast the trophozoites are much more sensitive to inactivation by chlorine and other disinfectants used to treat drinking water. A dose of chlorine of 1.0 mg/ liter with a free chlorine residual of 0.25 mg/ liter after 30 minutes resulted in a 99.99% reduction of trophozoites ( Cursons et al., 1980) of A. castellanii at pH 7.0 and 25 ° C. A similar reduction with a dose of chlorine dioxide of 2.9 mg/ liter ( 0.65 mg/ liter after 30 minutes) was achieved with chlorine dioxide, and an ozone dose of 6.75 mg/ liter ( residual 0.078 mg/ liter after 30 minutes). The experiments were conducted in distilled water. Thus, although the trophozoites are inactivated by these disinfectants, they are significantly more resistant than bacteria. The resistance of A. castellanii to chlorine has been shown to add to the resistance of Legionella pneumophila growing within the Acanthamoeba and may play a significant role in the survival of opportunistic bacteria and their ecology and persistence in distribution systems, cooling towers, hot tubs, and other environments. Kilvington and Price ( 1990) found that A. polyphaga were found to protect the legionellas from at least 50 mg/ liter of free chlorine. Control of Acanthamoeba in distribution systems may be necessary for control of Legionella pneumophila and Mycobacterium avium.

6.4 Dose Response Badenoch et al. ( 1990) demonstrated Acanthamoeba infections could be induced in the rat cornea by co inoculation with the bacterium Corynebacterium xerosis. The co inoculation with C. xerosis was necessary to induce the Acanthamoeba infection. Infection resulted in 7 of 24 rats that were exposed to 103 trophozoites and 1 in 10 animals when exposed to 104 trophozoites. At least 104 C. xerosis had to be co inoculated to achieve these infection rates. The results suggest that at least 103 trophozoites are necessary to cause Acanthamoeba eye infection.

6.5 Risk Characterization Acanthamoeba eye infections result from a combination of some eye trauma or contact lens use and other potential factors listed in Table 6.2. The concentration of Acanthamoeba in tapwater or aquatic environments may enhance the risk of infection ( Figure 6.1). Acanthamoeba infections in contact lens wearers can be eliminated by proper care of the lens to avoid exposure Health Effects Support Document for Acanthamoeba 6 4 Table 6.2 Mechanisms Involved in Acanthamoeba Keratitis ° Previous epithelial trauma ° Virulence of the organism ° Number of organisms ( on the contact lens, in the disinfection fluid, in the contaminated water ° Capability of the ameba to adhere to the cornea ° Duration of exposure ° Immune response ( presence of antibodies in tears)

to the organism. Exposure to contaminated water is the significant risk factor for contact lens wearers. Since Acanthamoeba cysts are resistant to inactivation by chlorine, a common disinfectant used for tapwater, exposure of the contact lens to tapwater should be avoided. Proper disinfection of contact lenses and the solutions they come into contact with is essential to prevent infection.

Acanthamoeba may also play a significant role in the potential for transmission of Legionella pneumophila and Mycobacterium avium via drinking water. The growth of these organisms within Acanthamoeba may provide protection from disinfectants and enhance their ability to cause disease in humans. Providing an unsuitable habitat for Achanthamoeba could potentially reduce these risks. Low organic matter and disinfectant residuals would be expected to minimize the number of bacteria upon which the amoeba feeds. This amoeba population may also be limited in size, but not necessarily eliminated by adequate disinfectant residuals.

While it is clear that a relationship exists between Acanthamoeba in water and keratitis, the role of tapwater is not clearly understood. Data on the occurrence and concentration of Acanthamoeba in the United States is lacking. One study suggests that municipal studies which may have become contaminated enhanced the risk of presumed Acanthamoeba keratitis ( Meier et al., 1998). Seasonal distribution of keratitis and abundance of Acanthamoeba in surface waters also suggests a relationship. Additional information on dose needed for infection and quantitative data on occurrence in drinking water supplies would help to better understand the potential risks to contact lens wearers and the general public. The incidence of recognized Acanthamoeba keratitis is around 1 2/ 106 ( Table 5.3). The highest incidence in the U. S., which may have been likened to flooding and the use of municipal water supplies, was 14/ 106 ( Meier et al., 1998). Even if all the cases of Acanthamoeba were associated with tapwater this would be Health Effects Support Document for Acanthamoeba 6 5 Health Effects Support Document for Acanthamoeba 6 6 less than the 1: 10,000 risk of infection per year that EPA has set as the goal for surface water supplies ( EPA, 1994; Regli et al., 1991). Health Effects Support Document for Acanthamoeba 7 1 7.0 ASSOCIATION OF CONTACT LENSES WITH ACANTHAMOEBIC KERATITIS 7.1 Types of Contact Lenses Contact lenses are worn on the surface of the eye to correct defects in an individual's vision. The first contact lens, made of glass, was developed in 1887 by Adolf Fick. The modern contact lens was developed in 1948, and is made of plastic and rests on a cushion of tears ( Table 7.1). It covers the cornea approximately over the iris and pupil. The hard plastic contact lenses had a limited wearing time because of potential irritation of the cornea. In the 1970' s, soft lenses, made from water absorbing plastic gel for greater flexibility, were introduced. In the 1980' s extended wear soft lenses, which can be worn without removal for several weeks at a time, were introduced. Soft contact lenses are usually more comfortable because they allow oxygen to penetrate to the surface of the eye. In the 1970' s gas permeable hard lenses ( which allow more oxygen to reach the eye) were developed.

The Food and Drug Administration must approve all contact lenses before they are available to the public. The types of contact lenses currently in use are listed in Table 7.2.

Table 7.1 History of Contact Lens Development1 Year Event 1887 First contact lens made from glass; covers the entire eye 1939 Contact lenses first made from plastic 1948 Plastic contact lenses designed to cover the cornea only 1971 Introduction of soft contact lenses 1978 Introduction of oxygen permeable lenses 1981 Food and Drug Administration approves soft contact lenses for extended ( overnight) wear 1986 Overnight wear oxygen permeable lenses become available 1987 Introduction of disposable soft contact lenses 1Source: Contact Lens Council, 2000 Health Effects Support Document for Acanthamoeba 7 2 Table 7.2 Types of Contact Lenses Type Comments Daily wear soft lenses Made of soft, flexible plastics that allow oxygen to pass through to the eye Cleaning is required Daily wear disposable soft lenses Typically no lens care is required Extended wear soft lenses Available for overnight wear Can usually be prescribed for up to seven days of wear without removal Extended wear disposable soft lenses Worn from one to six nights and then discarded Require little or no cleaning Rigid gas permeable lenses Made of slightly flexible plastics that allow oxygen to pass through to the eye Vision may be better than with soft lenses Long life ( 1 2 years) Daily and extended wear available 7.2 Demographics of Contact Lens Use Currently it is estimated that 34 million Americans wear contact lenses ( Contact Lens Council, 2000). Approximately 85% of the wearers use soft contact lenses and 15% use rigid gas permeable. Most wearers use daily wear lenses which are removed at bedtime, while 25% use extended wear lenses ( Table 7.3).

Extended wear lenses may be worn overnight and, in some cases, up to a week, before removal. Only 13% of contact lens wearers are 17 years of age or younger ( Table 7.4). Most soft contact lenses ( 45%) are worn by persons 26 to 39 years of age. In contrast, most rigid gas permeable lenses are worn by persons 40 years and older. Health Effects Support Document for Acanthamoeba 7 3 Table 7.3 Wearers and Types of Contact Lenses1 Type of lens Percent of wearers Soft lenses 85 Rigid gas permeable 15 Daily wear 75 Extended wear 25 1Source: Contact Lens Council Table 7.4 Age Distribution of Contact Lens Wearers in the United States1 Age ( years) % of soft contact lens wearers % of rigid gas permeable contact lens wearers < 17 10 3 18 to 25 23 10 26 to 39 45 26 $ 40 22 61 1 Source: Contact Lens Council, 2000 7.3 Risk Factors The use of contact lenses is the risk factor most commonly associated with acanthamoebic keratitis ( Table 7.5). Stehr Green et al. ( 1987) reported that 85% of the cases were associated with persons who wore contact lenses.

All types of contact lenses have been associated with acanthamoebic keratitis ( Table 7.6). Infection results from exposure to contaminated fluids used to wet the contact lens before placement on the eye or the use of contaminated fluids in storage cases. Any contact lens is a potential carrier of Acanthamoeba to the eye surface after being exposed to a contaminated fluid. Health Effects Support Document for Acanthamoeba 7 4 Table 7.5 Risk Factors A ssociated w ith Acanthamoebic Keratitis Risk Factor % of Acanthamoebic keratitis cases Wore contact lenses 85 Wore daily wear lenses 56 Wore extended wear lenses 19 History of corneal trauma 26 History of exposure to contaminated tapwater 25 Table 7.6 Type s of Contact Lenses Assoc iated w ith Acanthamoebic Keratitis Type of contact lens Percentage of cases Illingworth et al., 1995 Stehr Green et al., 1987 Moore et al., 1985 Daily wear soft 21 56 75 Daily wear 67

disposable soft Extended wear  19 14 Hard 8 2 6 Rigid gas permeable 4 7 4 The use of non sterile solutions such as tapwater, bottled water and non sterile distilled water have been associated with Acanthamoeba infections among contact lens wearers ( Moore et al., 1985; Stehr Green et al., 1987).

Infection is also associated with wearing contact lenses during swimming ( Stehr Green et al., 1987), use of hot tubs or exposure to natural springs ( Wilhemus and Jones, 1991). In a casecontrol study ( MMWR, 1987) it was found that of individuals who developed keratitis, 17 of 27 ( 63%) wore lenses while swimming, while 24 of 81 ( 30%) did not. Also, patients with keratitis Health Effects Support Document for Acanthamoeba 7 5 Table 7.7 Risk Factors for Acanthamoebic Keratitis in Contact Lens Wearers Risk Factor Use of tapwater to wet or store lenses Use of bottled water to wet or store lenses Use of distilled water to wet or store lenses Use of non sterile solutions to wet or store lenses Wearing lenses during swimming Wearing lenses in hot tubs Wearing lenses in natural springs Use of chlorine to disinfect lenses between uses Wetting lenses with saliva were more likely to wet lenses with saliva or wear lenses in a hot tub. The type of disinfectant used to treat the lenses during storage may also affect the risk of keratitis. Chlorine is not an effective means of disinfection and results in a greater risk of keratitis because of Acanthamoeba resistance to this disinfectant ( Illingworth et al., 1995).

7.4 Contact Lens Disinfection 7.4.1 Studies of Lens Disinfection Procedures for disinfecting different types of contact lenses and lens equipment have been investigated ( Knoll, 1971). Newer and safer methods for lens care were proposed by the U. S. Food and Drug Administration ( 1973) even before contact lens associated amoebic keratitis was discovered. Busschaert et al. ( 1978) had found that moist heat sterilization, 80 ° C for 10 minutes, provided an adequate margin of safety for disinfecting hydrophilic contact lenses. Acanthamoeba readily adheres to contact lenses. The degree of adherence depends on water content, surface tension and surface charge ( Gorlin et al., 1996). Kilvington ( 1989) investigated the killing capacity of moist heat against cysts of A. polyphaga, which survived a contact time of 60 minutes at 50 ° C to 60 ° C; but were inactivated when temperature was increased to 65 ° C to 70 ° C. However, when the experimental protocol was tested on lens cases of three patients who used moist heat, not all of the cysts were killed. This study suggested that even when lens cases are cleaned periodically, they probably should be replaced at some frequency to avoid a build up of debris and contaminating microorganisms. Health Effects Support Document for Acanthamoeba 7 6 Brandt et al. ( 1989) tested saline solutions, cleaning solutions, and disinfection solutions against three species of Acanthamoeba recovered from contact lens cases, i. e., A. castellanii, A. culbertsoni, and A. polyphaga. Although solutions containing hydrogen peroxide were the most effective, cysts were detected in all solutions for at least 6 hours after treatment. The authors concluded that, at the time of their study, none of the solutions available on the market were effective for eliminating cysts of Acanthamoeba within a short period of disinfection. Silvany et al. ( 1990) tested A. castellanii ATCC 30868 and A. polyphaga ATCC 30873 against 13 commercially available solutions. Growth occurred within as few as 30 minutes after exposure to one solution, with growth inhibited for up to 24 hours with five others. Two solutions containing hydrogen peroxide and three containing chlorohexidine inhibited growth within 30 minutes; one solution containing benzalkonium chloride inhibited growth within 1 hour. In this study and others ( Brandt et al., 1989), it was concluded that, at that time, there was neither one solution nor one treatment protocol that was effective against all species of Acanthamoeba. Rutherford et al. ( 1991) tested chlorhexidine in tablet form to find a procedure that would require less time for cleaning and disinfection. They tested a tablet dissolved in potable water for amoebicidal activity against trophozoites and cysts of A. castellanii and A. polyphaga isolated from human corneas, and against A. castellanii ATCC 30010. None of the amoebae excysted and grew after exposure times of 4, 6, 8, and 24 hours. Results showed that soft contact lenses could be successfully disinfected using tablets and non sterile tap water. The authors emphasized the fact that water used in this study came from the city of Cleveland, and that water used in other locales should be tested on an individual basis. Kilvington et al. ( 1991) compared three solutions for their ability to kill cysts of A. castellanii and A. polyphaga: hydrogen peroxide at 0.5, 1.0, and 3.0 percent, chlorhexadine gluconate at 0.004 percent, and thimerosal at 0.0025 percent strength. The assay procedures used in this study showed that hydrogen peroxide at three concentrations and chlorheximide gluconate killed the amoebae while thimerosal at the concentration use did not. Although chlorheximide inactivated 1x106 cysts down to approximately 1x101 within 4 hours, it was suggested that, although this exposure time was adequate, overnight disinfection probably would be safer.

7.4.2 Hydrogen Peroxide Hydrogen peroxide is the most effective chemical disinfectant against bacteria and Acanthamoeba, including trophozoites and cysts. It acts by oxidizing the organism ( Silvany et al., 1990). Hydrogen peroxide does not remove protein from the lens. This requires a separate cleaning process with a separate cleaning solution. Unneutralized hydrogen peroxide carried onto the cornea with the lens causes an acutely painful red eye with sterile inflammatory corneal infiltrates occurring due to oxidative damage to the epithelial surface. Neutralization is best performed after overnight wear in a vented storage case to release liberated oxygen; use of a nonvented case has resulted in serious ocular trauma from explosive propulsion of the lid into the eye. Because some lens wearers forget to neutralize the solution in the storage case in the morning, a one step product has been produced, based on adding a neutralizing tablet to the Health Effects Support Document for Acanthamoeba 7 7 storage case when the lenses are placed in the case for disinfection. The problem with these products so far has been the rapid neutralization of the hydrogen peroxide ( after 10 minutes). This is insufficient time to kill microbes on the lens.

7.4.3 Multi Purpose Solutions Due to problems with hydrogen peroxide, multi purpose solutions have been produced to clean and store lenses with a single solution without the need for neutralization. This is achieved by combining a poloxomer ( detergent) with a chemical disinfectant ( PHMB) or polyquaternium with appropriate buffers and EDTA. It is provided as a sterile solution in sufficient quantity for rub and rinse cleaning and storing of the lenses and washing of the storage case. Products may contain from 0.5 to 5ppm of PHMB. The lower concentration is less effective against bacteria and has no activity against Acanthamoeba. At this low concentration, eradicating Acanthamoeba depends on cleaning by the rinse and rub technique. The higher concentration is most effective against bacteria and fungi and is also acanthamoebicidal for 102 cysts ( Seal et al., 1992). Similarly, polyquaternium is used at low concentrations that have poor bactericidal activity and no acanthamoebicidal activity. Multipurpose solutions provide the easiest technique for the lens wearer to clean and disinfect the lens, and give better compliance results. The main advantage of these solutions is that the product is sterile, and there is no need to wash the storage case with tap water. The poloxomers used have a good surfactant action for removal of microbes adhering to the lens. Provided the storage case is changed monthly and tap water contamination is avoided, these solutions represent the most user friendly method. Bactericidal activity is reasonable, but not the best. Use of solutions with PHMB as the disinfectant at a minimum concentration of 5 ppm gives an enhanced microbiocidal effect, including activity against Acanthamoeba.

Hiti et al., 2001 recently reported the use of microwaves to inactivate contact lenses contaminated with acanthamoeba. Different types of contact lens cases were contaminated with trophozoites and cysts of three different Acanthamoeba species ( A. comandoni, A, castellanii, and A. hatchetti) and were exposed to microwave irradiation for various periods of time. 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. Health Effects Support Document for Acanthamoeba 8 1 8.0 DATA GAPS Risk from Acanthamoeba keratitis is complex depending upon the virulence of the particular strain, exposure, trauma or other stress to the eye and host immune response. Bacterial endosymbionts may also play a factor in pathogenicity of Acanthamoeba. Which factor( s) may be the most important is not clear. The recent work of Alizadeh et al., ( 2001) suggests that the ability of the host to produce IgA antibodies may be a significant factor. Thus, immune response could be a deciding factor as it appears in GAE infection and AIDS patients. If so then a certain sub population with an inability to produce IgA in the tears may be at greatest risk.

No data could be found on the occurrence or types of Acanthamoeba in tapwater in the United States. Published work on presence in tapwater does not provide information on the type of treatment the water received or the level of residual chlorine. Assessment of the pathogenicity by cell culture and molecular methods of Acanthamoeba in tapwater would also be useful in the risk assessment process for drinking water.

The possibility that Acanthamoeba spp. might serve as vectors for bacterial infections from water sources also needs to be explored. The bacterial endosymbionts include an interesting array of pathogens including Vibrio cholerae and Legionella pneumophila, both of which are well recognized water borne/ water based pathogens. Work is needed to determine if control of Acanthamoeba spp. is needed to control water based pathogens in water supplies.

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