United States
Environmental Protection
Agency
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
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
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 re viewers:
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, Walt Jay
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.
11
-------
Health Effects Support Document for Acanthamoeba
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
4.1.1.1 Freshwaters 4-2
4.1.1.2 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
111
-------
Health Effects Support Document for Acanthamoeba
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
IV
-------
Health Effects Support Document for Acanthamoeba
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
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
VI
-------
Health Effects Support Document for Acanthamoeba
Amphizoic amoeba
Anterior uveitis
Axenic
GLOSSARY OF TERMS
Amoeba able to live both free in nature and as pathogens in a host
Inflammation of the iris and ciliary body
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
Cornea
Endocyst
Endosymbiosis
Epithelium
Exocyst
Free-living
Granulomatous
amoebic encephalitis
Microscopy using a laser-scanning fluorescent microscope which gives
a digital two-dimensional signal that is reconstructed into a three
dimensional image
The clear, transparent anterior portion of the fibrous coat of the eye
The innermost cellulose-containing layer of the Acanthamoeba cyst. It
may be stellate, polygonal, oval, triangular, or round.
One organism living within the other in a mutually beneficial
relationship
The layer of cells forming the epidermis of the skin and the surface
layer of mucous and serous membranes
The wrinkled proteinaceous outer layer of the Acanthamoeba cyst
Replicate in the environment and do not require a host
Subacute opportunistic infection caused by Acanthamoeba spp.
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
IgG
The predominant antibody class present in secretions
The predominant antibody present in human serum
vn
-------
Health Effects Support Document for Acanthamoeba
Macrophage
Meningoencephalitis
Nodular scleritis
Ocular
Phagocytosis
Ring infiltrate
Sclera
Scleritis
Stroma
Stromal
Subacute
Uvea
Cells found in the body having the ability to engulf or phagocytose
particulate substances (e.g. bacteria)
Inflammation of the brain and meninges
A small aggregation of cells causing inflammation of the sclera
Concerning the eye or vision
Ingestion (engulfment) and digestion of bacteria
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.
A tough, white, fibrous tissue that covers the so-called white of the
eye, extending from the optic nerve to the cornea
Superficial and deep inflammation of the sclera
Foundation supporting tissues of an organ
Concerning or resembling the stroma of an organ
Between acute and chronic
The second vascular coat of the eye, lying immediately beneath the
sclera. It consists of iris, ciliary body, and choroid.
vin
-------
Health Effects Support Document for Acanthamoeba
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 included, astronyxis, A. castellanii, A. culbertsoni, A. divionensis, A. griffini, A. heatyi, 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.
1-1
-------
Health Effects Support Document for Acanthamoeba
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 sulfate-
gramicidin, 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
1-2
-------
Health Effects Support Document for Acanthamoeba
and Acanthamoeba, including trophozoites and cysts. Chlorine is not considered effective. Multi-
purpose 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 polyquateniium 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 bio film 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 maybe related to
concentrations of the organism present in surface waters and tapwater.
Granule mat ous 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 maybe 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
1-3
-------
Health Effects Support Document for Acanthamoeba
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.
1-4
-------
Health Effects Support Document for Acanthamoeba
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 included, 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
Flagellate
Acanthamoeba
Naegleria
Entamoeba hystolytica
Giardia lamblia
Apicomplexan Toxoplasma gondii
Cryptosporidium
Cyclospora
cayetanesis
eye infection (keratitis),
brain infection(meningo-encephalitis)
brain infection(meningo-encephalitis)
amoebic diarrhea (liver abscess)
diarrhea
fever, loss of fetus
diarrhea
diarrhea
2-1
-------
Health Effects Support Document for Acanthamoeba
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. (195 8) 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 Currently Identified Species 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
3-1
-------
Health Effects Support Document for Acanthamoeba
Table 3.2 Acanthamoeba Species Classification (Pussard and Pons, 1977)
Group I
A. astronyxis
A. comandoni
A. echinulata
Group II
A. castellani
A. mauritaniensis
A. polyphaga
A. lugdunesis
A. quina
A. rhysodes
A. divionensis
A. paradivionensis
A. griffini
A. triangularis
Group III
A. palastinensis
A. culbertsoni
A. lenticulata
A. pustulosa
A. royreba
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 |jm 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 |jm 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 maybe of importance
in the persistence and spread of these organisms in the environment (King et al, 1988).
3-2
-------
Health Effects Support Document for Acanthamoeba
Figure 3.1 Life Cycle of Acanthamoeba Species
Vegetative form or tr ophozoite
Reproduction by
binary fission with
dissolution of nuclear
membrane at prophase
.
Encystment
3-3
-------
Health Effects Support Document for Acanthamoeba
Figure 3.2 Acanthamoeba Trophozoite (amebic stage). Note the characteristic spine-
like acanthapodia. (Visvesvara, 1987)
i/
Figure 3.3 Cysts of Acanthamoeba. Note the characteristic double wall with an
outerwrinkled ectocyst and an inner polygonal endocyst (Visvesvara, unpublished)
3-4
-------
Health Effects Support Document for Acanthamoeba
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
Klebsiellapneumoniae (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 maybe 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°C to 45°C, or higher.
3.5 Significance 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 (Bowling et al., 1992).
It may also be involved in the observed phenomenon thatZ. pneumophila can be viable but non-
detectable by cultivation on agar-based systems (Connor et al., 1993). Hay and Seal (1994b)
-------
Health Effects Support Document for Acanthamoeba
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 Endosymbionts* 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
3-6
-------
Health Effects Support Document for Acanthamoeba
Figure 3.4 Significance of Endosymbiosis to Waterborne Disease Transmission
Amoeba
Bacteria
Enhanced virulence
of both organisms
Endo symbiotic
relationship develops
Enhanced r esistaiice
of bacteria to
disinfectants
3-7
-------
Health Effects Support Document for Acanthamoeba
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
Tap water (Mexico)
Bottled water (Mexico)
Hospital tap water
Eyewash stations
Freshwater ponds
Thermal water
Well water
Physiotherapy tubs
Aquaria
Municipal sewage
Ocean sewage dump site
House dust
Garden soil
Sand box
Garden vegetables
Fish
Air conditioner
CrespoetaL, 1990
Riverae? al., 1979
Rivera ef al, 1981
RohretaL, 1998
Tyndalle^a/. , 1987
John and Howard, 1995
DeJonckheere, 1979, Dive et al, 1982
Jones et al., 1975
Penas-Ares et al., 1994
DeJonckheere, 1979
Singh and Das, 1972
Sawyere? al, 1982
Yamaurae^a/., 1993
Singh, 1952
Yamaurae^a/., 1993
Rude et al., 1984
Taylor, 1977
Walker ef al, 1986
4-1
-------
Health Effects Support Document for Acanthamoeba
4.1 Water
4.1.1 Surface Waters
4.1.1.1 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°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 750 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, B.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 fromponds 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.
4.1.1.2 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^ al. (1992) recovered several species of Acanthamoeba from sewage-
contaminated inshore New York and New Jersey shellfish beds that periodically were closed to
4-2
-------
Health Effects Support Document for Acanthamoeba
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 castellaniiwerQ
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 anew 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.
4-3
-------
Health Effects Support Document for Acanthamoeba
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 "HartmanneHa-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 (Janitscnke 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°C to 45°C. Two strains were
identified as A castellanti, one as A lugdunensis and the others as Acanthamoeba spp. All were
pathogenic to mice. The authors conducted a second study (Rivera^ 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°C to 64°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.
4-4
-------
Health Effects Support Document for Acanthamoeba
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 follow-
up 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, Dust and Soil
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^, 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. castellaniiper 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
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.
4-6
-------
Health Effects Support Document for Acanthamoeba
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 Clinical and Pathological Features of Granulomatous
Amoebic Encephalitis (GAE) and Acanthamoeba Keratitis (AK)
Features
GAE
AK
Predisposing Factors
Epidemiology
Usual Portals of Entry
Incubation Period
Clinical Course
Prognosis
Immunodeficiency; AIDS;
Debilitating chronic disease
Worldwide
Lungs; skin; nose;
neuroepithelium
Probably weeks to months
Subacute or chronic (several
weeks to months);
Almost always fatal
Clinical Symptoms and Signs Personality changes;
confusion; seizures; nausea;
headache; dizziness
Treatment
Itraconazole; Miconazole;
Sulfametazine;
Pentamididine IV (in vitro)
Good health, corneal trauma,
contaminated contact lens
wearing
Worldwide
Corneal abrasion
Probably days
Subacute or chronic
Good if properly treated
Eye pain; typical corneal ring
"infiltrate"; photophobia;
blurred vision
Polyhexamethylene
biguamide; Propamidine
isethionate
5-1
-------
Health Effects Support Document for Acanthamoeba
Figure 5.1 Life cycle of Acanthamoeba spp. and Human Infection
Eff
n
I*
.X"
5-2
-------
Health Effects Support Document for Acanthamoeba
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 (Acanthamoebic 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 Symptoms of Patients
with Acanthamoeba Keratitis
•Young, healthy individuals
• Soft contact lens wearers
• Non-preserved or non-sterile solution used for
storage of contact lens
• Eye trauma
• Usually one eye affected
• Extreme eye pain
• Corneal breakdown of the epithelial
• 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
5-3
-------
Health Effects Support Document for Acanthamoeba
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 a/.(1987) reported that most patients (95%) had at
least one risk factor for acanthamoebic keratitis, the 85% who wore contact lenses, most wore
5-4
-------
Health Effects Support Document for Acanthamoeba
daily wear (56%) or extended wear soft (19%). Some patients (including both contact lens
wearers) (26%) had a history of cornea! 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.
Figure 5.2 Slit lamp view showing a paracentral complete ring infiltrate of the
cornea. The ring infiltrate is diagnostic of Acanthamoeba infections
(Theodore et al., 1985)
Figure 5.3 Normal eye
5-5
-------
Health Effects Support Document for Acanthamoeba
5.1.2 Diagnosis of Acanthamoeba Keratitis
While positive diagnosis of acanthamoebic keratitis can be made by in vivo con focal microscopy,
diagnostic tests usually rely on demonstrating amoebae on corneal scrapings or biopsy material
(Seal et a/., 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 wiihE. 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 well-
known 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
5-6
-------
Health Effects Support Document for Acanthamoeba
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 (Picker, 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 sulfate-
polymixin 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).
5-7
-------
Health Effects Support Document for Acanthamoeba
Table 5.3 Worldwide Incidence of Acanthamoeba Keratitis
Incidence per
1,000,000
Population
Country
Year(s)
Reference
1.65 to 2.01
1.1
149
0.58 to 0.71
1.40
1.30
Contact Lens
Wearer (CLW)
CLW
CLW
General
Population (GP)
GP
GP - Iowa well
water
USA
Netherlands
UK
USA
UK
USA
1985-1987
1996
1996
1985-1987
1996
1993-1994
Schaumberg etal.,
1998
Cheng et al, 1999
Seal, 2000
Schaumberg etal.,
1998
Radforde^a/., 1998
Meier ef 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 non-
pathogenic 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
5-8
-------
Health Effects Support Document for Acanthamoeba
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 thai 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.
5-9
-------
Health Effects Support Document for Acanthamoeba
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 Encephalitis
Granule mat ous 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.
5-10
-------
Health Effects Support Document for Acanthamoeba
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).
Damaged Section of Brain
Balamuthia has been identified in approximately 40 patients in the United States (U.S.), including
>10 with ADDS infection (Martinez et a/., 1997, Visvaresvara, 2001). In contrast, Acanthamoeba
has accounted for approximately 84 (-50 with ADDS) cases in the U.S. and 120 worldwide
(Martinez et a/., 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
a/., 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 a/., 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
5-11
-------
Health Effects Support Document for Acanthamoeba
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-fiuorocytosine maybe
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 pep tide 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-Cabrale^a/., 2000; EPA, 1998).
5-12
-------
Health Effects Support Document for Acanthamoeba
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 neuroepithehum 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 Animals and Wildlife
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, DykovaetaL, 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
5-13
-------
Health Effects Support Document for Acanthamoeba
patient (Teknos et al., 2000) and possible association with intestinal disorders (Hoffler and
Rubel, 1974; Mehta and Guirges, 1979; Thampraserte^a/., 1993).
5.5 Immunocompromised Individuals
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).
5-14
-------
Health Effects Support Document for Acanthamoeba
5.7 Effect of Endosymbiosis on Virulence
Acanthamoeba spp. has been demonstrated to develop endosymbiotic relationships with a
number of waterborne bacteria, including Legionellapneumophila and Mycobacterium avium
(Table 3.3). This relationship maybe 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 ofM avium in
water environments occurs primarily within protozoa, the fact that M. avium has temperature-
dependant 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.
5-15
-------
Health Effects Support Document for Acanthamoeba
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.
6-1
-------
Health Effects Support Document for Acanthamoeba
Table 6.1 Human Infection Caused by Species of Acanthamoeba
Species of
Acanthamoeba
CNS Eye
infection infection
Other tissues
Reference
A. astronyxis
A. castellanii
X
X
X
A. culbersoni
X
X
A. divionensis X
A. griffini
A. hatchetti
A. healyi X
A. palestinensis X
A. polyphaga
A. rhysodes X
X
X
X
X
Adrenal, lymph
node, sinus, skin,
thyroid
Lung, prostate,
bone, muscle,
sinus, skin
Liver, spleen,
uterus, skin
Gulletteffl/. (1979)
Martinez (1982)
Martinez et al. (1977)
Moore ef al. (1985)
Borochovitz etal. (1981)
Gonzalez ef a/. (1986)
Martinez et al. (1977)
Wiley etal. (1987)
Mannish al. (1986)
May e£ a/. (1992)
DiGregorio (1992)
Ledeee^a/. (1996)
Cohen ef al. (1985)
Kim e£ a/. (2000)
Ofori-Kwakye et al. (1986)
Singh and Petri (2000)
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 um) and
cysts (15 to 28 um) 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
6-2
-------
Health Effects Support Document for Acanthamoeba
iodine. The chlorine resistance of two different strains varied considerably. A 99.99% (4 Iog10)
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 Iog10) inactivation of the cysts. The
viability of the cysts was detected with a plaque assay on a lawn of Escherichia co/z 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 ofLegionella 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 Corynebacteriumxerosis. 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
6-3
-------
Health Effects Support Document for Acanthamoeba
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 ofLegionella
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
a/., 1998). Seasonal distribution of keratitis and abundance ofAcanthamoeba 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
a/., 1998). Even if all the cases of Acanthamoeba were associated with tapwater this would be
-------
Health Effects Support Document for Acanthamoeba
Figure 6.1 Eye Trauma and Contact Lenses as Determinants of Susceptibility to
Acanthamoeba Keratitis
High bacterial
numbers
Warm
temperatures
Seasonal peaks
in surface waters
Resistance to
disinfectants
Increased susceptibility
to infection
Contact lens wearer
Use of non-sterile
wetting solutions
Use of tapwater
as a wetting or
storage solution
Physical injury
Work related
eye irritation
Several conditions, such as use of tapwater as a wetting solution, can
increase exposure to Acanthamoeba. Individuals who wear contact
lenses or have experienced eye trauma are at greater risk to
Acanthamoeba infections.
6-5
-------
Health Effects Support Document for Acanthamoeba
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).
6-6
-------
Health Effects Support Document for Acanthamoeba
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 Pick. 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
1 Source: Contact Lens Council, 2000
7-1
-------
Health Effects Support Document for Acanthamoeba
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.
7-2
-------
Health Effects Support Document for Acanthamoeba
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
'Source: 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.
7-3
-------
Health Effects Support Document for Acanthamoeba
Table 7.5 Risk Factors Associated with 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 Types of Contact Lenses Associated with Acanthamoebic Keratitis
Type of
contact lens
Daily wear soft
Daily wear
disposable soft
Extended wear
Hard
Rigid gas permeable
Illingworth et al, 1995
21
67
-
8
4
Percentage of cases
Stehr-Green et al.,
1987
56
-
19
2
7
Moored al., 1985
75
-
14
6
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 case-
control 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
7-4
-------
Health Effects Support Document for Acanthamoeba
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 (Gorhnet 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.
7-5
-------
Health Effects Support Document for Acanthamoeba
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 pofyphaga. 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. pofyphaga 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. pofyphaga 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. pofyphaga: 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 non-
vented 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
7-6
-------
Health Effects Support Document for Acanthamoeba
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 a/., 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 a/., 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.
7-7
-------
Health Effects Support Document for Acanthamoeba
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 a/., (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 maybe 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.
Finally, better (i.e. greater range of concentration of cysts) dose response data in animals would
be useful to assess the probability of infection of susceptible individuals.
8-1
-------
Health Effects Support Document for Acanthamoeba
9.0 REFERENCES
Alizadeh, H., He, Y., McCulley, J.P., Ma, D., Stewart, G.L., Via, M., Haehling, E., and
Niederkorn, J. Y. 1995. Successful immunization against Acanthamoeba keratitis in a pig model.
Cornea 14:180-186.
Alizadeh, H., Apte, S., El-Agha, M.S., Li, L., Hurt, M., Howard, K., Cavanagh, H.D., McCulley,
J.P., and Niederkorn, J.Y. 2001. Tear IgA and serum IgG antibodies against Acanthamoeba in
patients with Acanthamoeba keratitis. Cornea 20:622-627.
Anderlini, P., Przepiorka, D., Luna, M., Langford, L, Andreeff, M., Claxton, D. and Deisseroth,
A.B. 1994. Acanthamoeba meningoencephalitis after bone marrow transplantation. Bone
Marrow Transplant. 14:459-461.
Asiri, S.M.B.A., Chinnis, R.J. andBanta, W.C. 1990. Potentially pathogenic species of
Acanthamoeba and Hartmannella (Protzoa:Amoebida) in sediment of the Potomac River near
Washington, D. C. J. Helminthol. Soc. Wash. 57:88-90
Ayers, K.M., Billups, A.H. and Garner, P.M. 1972. Acanthamoebiasis in a dog. Vet. Pathol.
9:221-226.
Badenoch, P.R. 1991. The pathogenesis of Acanthamoeba keratitis. Australian and New Zealand
J. Ophthalmol. 19:9-20.
Badenoch, P.R., Johnson, A.M., Christy, P.E., and Coster, D.J. 1990. Pathogenicity of
Acanthamoeba and a Corynebacterium in the. Arch. Ophthalmol. 108:107-112.
Bauer, R.W., Harrison, L.R., Watson, C.W., Styer, E.L., and Chapman, W.L., Jr. 1993. Isolation
of Acanthamoeba sp. from a greyhound with pneumonia and granulomatous amebic encephalitis.
J. Vet. Diagn. Invest. 5:386-391.
Berger, S.T., Mondino, B.J., Hoft, R.H., Donzis, P.B., Holland, G.N., Farley, M.K., and
Levenson, J.E. 1990. Successful medical management of Acanthamoeba keratitis. Am. J.
Ophthalmol. 110:395-403.
Booton, G.C., Dykova, L, Lorn, J., Schroeder-Diedrich, J.M. and Byers, T.J. 1999.
Morphological and rDNA similarities of Acanthamoeba strains parasitic in fish and those
causing human disease. J. Eukaryotic Microbiol. 46:6A.
9-1
-------
Health Effects Support Document for Acanthamoeba
Borochovitz, D., Martinez, A. J., and Patterson, G.T. 1981. Osteomyelitis of a bone graft of the
mandible with Acanthamoeba castellanii infection. Human Pathol. 12:573-576.
Bose, K., Ghosh, O.K., Ghosh K.N., Bhattacharya, A. and Das, S.R. 1990. Charcterization of
potentially pathogenic free-living amoebae in sewage samples of Calcutta, India. Brazilian J.
Med. Biol. Res. 23:1271-1278.
Bovee, B.C., Wilson, D.E., and Telford, S.R., Jr. 1961. Some amoebas and amebofiagellates
inquilinic in Florida reptiles. J. Protozool. 8:15.
Bowman, E.K., Vass, A.A., Mackowski, R., Owen, B.A., and Tyndall, R.L. 1996. Quantitation
of free-living amoebae and bacterial populations in eyewash stations relative to flushing
frequency. Am. Ind. Hyg. Assoc. J. 57:626-633.
Brandt, F.H., Ware, D.A. and Visvesvara, G.S. 1989. Viability of Acanthamoeba cysts in
ophthalmic solutions. Appl. Environ. Microbiol. 55:1144-1146.
Brasseur, G., Favennec, L., Perrine, D., Chenu, J.P., and Brasseur, P. 1994. Successful treatment
of Acanthamoeba keratitis by hexamidine. Cornea 13:459-462.
Brown, T.J. and Cursons, R.T.M. 1977. Pathogenic free-living amebae (PFLA) from frozen
swimming areas in Oslo, Norway. Scand. J. Infect. Dis. 9:237-240.
Brown, T.J., Cursons, R.T.M., Keys, E.A., Marks, M., and Miles, M. 1983. The occurrence and
distribution of pathogenic free-living amoebae in thermal areas of the North Island of New
Zealand. New Zealand J. Marine and Freshwater Res. 17:59-69.
Busschaert, S.C., Good, R.C. and Szabocsik, J. 1978. Evaluation of thermal disinfection
procedures for hydrophilic contact lenses. Appl. Environ. Microbiol. 35:618-621.
Butt, C.G. 1966. Primary amebic meningoencephalitis. N. Engl. J. Med 274:1473-1476.
Callicott, J.C., Nelson, E. C., Jones, M.M., dos Santos, J.G., Utz, J.P., Duma, R.J. and Morrison,
J.V., Jr. 1968. Meningoencephalitis due to pathogenic free-living amoebae. J.A.M.A. 206:579-
582.
Cerva, L. 1971. Studies of limax amoebae in a swimming pool. Hydrobiologia. 38:141-161.
9-2
-------
Health Effects Support Document for Acanthamoeba
Cerva, L. 1989. Acanthamoeba culbertsoni and Naegleriafowleri: occurrence of antibodies in
man. J. Hyg. Epidemiol. Microbiol. Immunol. 33:99-103.
Chang, J.C.H., Ossoff, S.F., Lobe, D.C., Dorfman, M.H., Dumais, C.M., Quails, R.G., and
Johnson, J.D. 1985. UV Inactivation of Pathogenic and Indicator Microorganisms. Appl. Env.
Micro. 49:1361-1365.
Cheng, K.H., Leung, S.L., Hoekman, H.W., Beekhuis, W.H., Mulder, P.G.H., and
Geerards,A.J.M. 1999. Incidence of contact-lens-associated microbial keratitis and its related
morbidity. The Lancet 354:181-185.
Chu, D.M., Miles, H.,Toney, D., Ngyuen, C. and Marciano-Cabral, F. 1998. Amebicidal activity
of plant extracts from Southeast Asia on Acanthamoeba spp. Parasitol. Res. 84:746-752.
Cirillo, J.D., Falkow, S., Tompkins, L.S., and Bermudez, L.E. 1997. Interaction of
Mycobacterium avium with Environmental Amoebae Enhances Virulence. Infect. Immun.
65:3759-3767.
Cirillo, J.D., Cirillo, S.L., Yan, L., Bermudez, L.E. Falkow, S., and Tompkins, L.S. 1999.
Intracellular growth in Acanthamoeba castellanii affects monocyte entry mechanisms and
enhances virulence of Legionellapneumophila. Infect. Immun. 67:4427-4434.
Cohen, E.J., Buchanan, H.W., Laughrea, P.A., Adams, C.P., Galentine, P.G., Visvesvara, G.S.,
Folberg, R. Arentsen, J.J., and Laibson, P.R. 1985. Diagnosis and management of Acanthamoeba
keratitis. Am. J. Ophthalmol. 100:389-395.
Connor, R., Hay, J., Mead, A.J.C., and Seal, D.V. 1993. Reversal of inhibitory effects of
Acanthamoeba castellanii lysate for Legionella pneumophila using catalase. J. Microbiol.
Methods 18:311-316.
Contact Lens Council. 2000. www.contactlenses.org.
Crespo, E.P., Mallen M.M., Monica, M.P., Ares, P., Fernandez, M.C.A., and Combarro, M.P.C.
1990. Isolation of amoebae of the genera Naegleria and Acanthamoeba from public fountains in
Galicia (N.W. Spain). Water, Air and Soil Pollution. 53:103-111.
Cruz, O.A., Sabir, S.M., Capo, H., Alfonso, B.C. 1993. Microbial Keratitis in Childhood.
Ophthalmology 100:192-196.
9-3
-------
Health Effects Support Document for Acanthamoeba
Culbertson, C.G., Smith, J.W., and Miner, J.R. 1958. Acanthamoeba: Observations on animal
pathogenicity. Science. 127:1506.
Culbertson, C.G. 1971. The pathogenicity of soil amebas. Annu. Rev. Microbiol. 25:231-254.
Cursons, R.T., Brown, T.J., and Keys, E.A. 1980. Effect of disinfectants on pathogenic free-
living amoebae: in axenic conditions. Appl. Environ. Microbiol. 40:62-66.
Cursons, R.T., Brown, T.J., Keys, E.A., Moriarty, K.M., and Till, D. 1980. Immunity to
pathogenic free-living amoebae: role of humoral antibody. Infect. Immun. 29:401-407.
Daggett, P.-M. 1982. Microbial Hazards of Diving in Polluted Waters - A Proceedings, pp. 39-
42, (R.R. Colwell, ed.). Univ. Md. Sea Grant Publ. No. UM-SG-TS-82-01, Univ. Md., College
Park, Md.
DAversa, G., Stem, G.A., and Driebe, W.T. Jr. 1995. Diagnosis and successful medical
treatment of Acanthamoeba keratitis. Arch. Ophthalmol. 113:1120-1123.
de Jonckheere, J., and Van de Voorde, H. 1976. Differences in Destruction of Cysts of
Pathogenic and Nonpathogenic Naegleria and Acanthamoeba by Chlorine. Appl. Environ.
Microbiol. 31:294-297.
de Jonckheere, J.F. 1979a. Pathogenic free-living amoebae in swimming pools: survey in
Belgium. Ann. Microbiol. (Inst. Pasteur). 130 8:205-212.
de Jonckheere, J.F. 1979b. Occurrence of Naegleria and Acanthamoeba in aquaria. Appl.
Environ. Microbiol. 38:590-593.
de Jonckheere, J.F. 1987. Taxonomy. In: Rondanelli, E.G., ed. Amphizoic Amoebae Human
Pathology. Padua. Italy: Piccin Nuova Libraria, 25-48.
de Jonckheere, J.F., and Michel, R. 1988. Species identification and virulence of Acanthamoeba
strains from human nasal mucosa. Parasitol. Res. 74:314-316.
Detterline, J.L., and Wilhelm, W.E. 1991. Survey of pathogenic Naegleria fowleri and
thermotolerant amebas in federal recreational waters. Trans. Am. Microsc. Soc. 110:244-261.
9-4
-------
Health Effects Support Document for Acanthamoeba
Di Gregorio, C., Rivasi, F., Mongiardo, N., De Rienzo, B., Wallace, S., and Visvesvara, G.S.
1992. Acanthamoeba meningoencephalitis in a patient with acquired immunodeficiency
syndrome. Arch. Pathol. Lab. Med. 116:1363-1365.
Dive, D., Delattre, J.M., and Leclerc, H. 1982. Occurrence of thermotolerant amoebae in an
electric power plant cooling pond. J. Therm. Biol. 7:11-14.
Douglas, M. 1930. Notes on the classification of the amoeba found by Castellani in cultures of a
yeast-like fungus. J. Trop. Med. Hyg. 33:258-259.
Dowling, J.N., Saba, A.K., and Glew, R.H. 1992. Virulence factors of the family Legionellacae.
Microbiol. Rev. 56:32-60.
Dwivedi, J.N. and Singh, C.M. 1965. Pulmonary lesions in an Indian buffalo associated with
Acanthamoeba sp. Indian J. Microbiol. 5:31-34.
Dykova, I., Lorn, J., Machackov4 B. and Sawyer, T.K. 1996. Amoebic infections in goldfishes
and granulomatous lesions. Folia Parasitol. 43:81-90.
EPA. Environmental Protection Agency. 1994. Enhanced Surface Water Treatment Rule.
Federal Register 59 FR 38832, July 29, 1994.
EPA. Environmental Protection Agency. 1998. Demographic Distribution of Sensitive
Population Groups. Office of Science and Technology, Human Ecological Effects Division,
Washington, D.C.
Ferrante, A. 1991. Free-living amoebae: pathogenicity and immunity. Parasite Immunol. 13:31-
47.
Picker, L. 1988. Acanthamoeba keratitis - the quest for a better prognosis. Eye 2 Suppl :S37- 45.
Fields, B.S., Sanders, G.N., and Barbaree, J.M. et al. 1989. Intracellular multiplication of
Legionellapneumophila in amoebae isolated from hospital hot water baths. Curr. Microbiol.
18:131-137.
Fields, B.S. 1993. Legionella and protozoa: interaction of a pathogen and its natural host. In:
Barbaree, J.M., Breiman, R.F. Dufour, A.P. (eds.) Legionella current status and emerging
perspectives. Washington, D.C. American Society for Microbiology 129.
9-5
-------
Health Effects Support Document for Acanthamoeba
Franke, E.D., and Mackiewicz, S. 1982. Isolation of Acanthamoeba andNaegleria from the
intestinal contents of freshwater fishes and their potential pathogenicity. J. Parasitol. 68:164-166.
Friedland, L.R., Raphael, S.A., Deutsch, E.S., Johal, J., Martyn, L.J., Visvesvara, G.S., and
Lischner, H.W. 1992. Disseminated Acanthamoeaba infection in a child with symptomatic
human immunodeficiency virus infection. Pediatric Inf. Dis. J. 11:404-407.
Fritsche, T.R., Sobek, D., and Gautom, R.K. 1998. Enhancement of in vitro cytopathogenicityby
Acanthamoeba spp. following acquisition of bacterial endosymbionts. FEMS Microbiology
Letters 166:231-236.
Fritsche, T.R., Horn, M., Seyedirashsti, S., Gautom, R., Schleifer, K.-H., and Wagner, M. 1999.
In situ detection of novel bacterial endosymbionts of Acanthamoeba spp. phylo genetically
related to members of the Order Rickettsiales. Appl. Environ. Microbiol. 65:206-212.
Gardner, H.A., Martinez, A.J., Visvesvara, G.S., and Sotrel, A. 1991. Granulomatous amebic
encephalitis in an AIDS patient. Neurology 41:1993-1995.
Gogate, A., Singh, B.N., Deodhar, L.P., and Jhala, H.I. 1984. Primary amoebic meningo-
encephalitis caused by Acanthameoba. J. Postgrad. Med. 30:125-128.
Gonzalez, M.M., Gould, E., Dickinson, G., Martinez, A.J., Visvesvara, G., Cleary, T.J., and
Hensley, G.T. 1986. Acquired immunodeficiency syndrome associated with Acanthamoeba
infection and other opportunistic organisms. Arch. Path. Lab. Med. 110:749-751
Gordon, S.M., Steinberg, J.P., DuPuis, M.H., Kozarsky, P.E., Nickerson, J.F., and Visvesvara,
G.S. 1992. Culture isolation of Acanthamoeba species and leptomyxid amebas from patients
with amebic meningoencephalitis, including two patients with AIDS. Clin. Inf. Diseases,
15:1024-1030.
Gorlin, A.I., Gabriel, M.M., Wilson, L.A. and Ahearn, D.G. 1996. Binding of Acanthamoeba to
hydrogel contact lenses. Current Eye Res. 15:151-155.
Griffin, J.L. 1972. Temperature tolerance of pathogenic and nonpathogenic free-living amoebas.
Science. 178:869-870.
Gullett, J., Mills, J., Hadley, K., Podemski, B., Pitts, L., and Gelber, R. 1979. Disseminated
gramilomatous Acanthamoeba infection presenting as an unusual skin lesion. Am. J. Med.
67:891-896.
9-6
-------
Health Effects Support Document for Acanthamoeba
Hamadto, H.H., Aufy, S.M., El-Hayawan, LA., Saleh, M.H., andNagaty, I.M. 1993. Study of
free living amoebae in Egypt. J. Egyptian Soc. Parasitol., 23:631-637.
Hamburg, A., and De Jonckheere, J.F. 1980. Amoebic keratitis. Ophthalmologica 181:74-80.
Hay, J., Seal, D.V., Billcliffe, B., Freer, J.H. 1995. Non-cuhurablQLegioneUapneumophila
associated with Acanthamoeba castellanii detection of the bacterium using DNA amplification
and hybridization. J. Appl. Bateriol. 78:61-65.
Hay, J., and Seal, D.V. 1994a. Surveying for legionnaire's disease bacterium. Curr. Opin. Infect.
Dis. 7:479-483.
Hay, J. and Seal, D.V. 1994b. Monitoring of hospital water supplies for Legionella. J. Hosp.
Infect. 26:75-78.
Hiti, K., Walochnik, J., Faschinger, C., Haller-Schober, E.M., and Aspock, H. 2001. Microwave
treatment of contact lens cases contaminated with acanthamoeba. Cornea 20:467-470.
Hoffler, A.S., and Rubel, L.R. 1974. Free-living amoebae identified by cytologic examination of
gastrointestinal washings. Acta Cytol. 18:59-61.
Houang, E., Lam, D., Fan, D., and Seal, D. 2001. Microbial keratitis inHong Kong: relationship
to climate, environment and contact-lens disinfection. Trans. R. Soc. Trop. Med. Hyg. 95:361-
367.
Illingworth, C.D., Cook, C.D., Karabatsas, C.H., and Easty, D.L. 1995. Acanthamoeba keratitis:
risk factors and outcome. Br. J. Ophthalmol. 79:1078-1082.
Illingworth, C.D., and Cook, S.D. 1998. Acanthamoeba keratitis. Surv. Ophthalmol. 42:493-
508.
Ishibashi, Y.,Matsumoto, Y., Kabata, T., Watanabe, R., Hommura, S., Yasuraoka, K., andlshii,
K. 1990. Oral itraconazole and topical miconazole with debridement for Acanthamoeba keratitis.
Am. J. Ophthalmol. 109:121-126.
Jadin, J.-B., Willaert E., and Hermanne, J. 1973. Presence d'amibes limax dans I'mtestin de
1'homme et des animaux. Academic Royale des Sciences D'outre Mer. Bull. Seances 1973-3,
Bruxelles, 520-526.
9-7
-------
Health Effects Support Document for Acanthamoeba
Jahnes, W.G., Fullmer, H.M., and Li, C.P. 1957. Free living amoebae as contaminants in
monkey kidney tissue culture. Soc. Exp. Biol. Med. 96:484-488.
Janitschke, K., Werner, H., and Muller, G. 1980. Examinations on the occurrence of free-living
amoebae with possible pathogenic traits in swimming pools. Zbl. Bakt. I. Abt. Orig. B. 170:108-
122.
Janitschke, K., Martinez, A.J., Visvesvara, G.S., and Schuster, F. 1996. Animal model
Balamuthia mandrillaris CNS infection: contrast and comparison in immunodeficient and
immunocompetent mice: a murine model of "granulomatous" amebic encephalitis. J.
Neuropathol. Exp. Neurol. 55:815-821.
John, D. T., and Howard, M.J. 1995. Seasonal distribution of pathogenic free-living amebae in
Oklahoma waters. Parasitol. Res. 81:193-201.
Johns, K.J., O'Day, D.M., and Feman, S.S. 1988. Chorioretinitis in the contralateral eye of a
patient with Acanthamoeba keratitis. Ophthalmology 95:635-639.
Jones, B.R., McGill, J.I., and Steele, A.D.McG. 1975. Recurrent suppurative kerato-uveitis with
loss of eye due to infection by Acanthamoeba castellanii. Trans. Ophthalmol. Soc. U.K. 95:210-
213.
Jones, D.B., Visvesvara, G.S., and Robinson, N.M. 1975. Acanthamoebapotyphaga keratitis and
Acanthamoeba uveitits associated with fatal menengoencephalitis. Trans. Ophthalmol. Soc. U.K.
95:221-232.
Kadlec, V. 1978. The occurrence of amphizoic amebae in domestic animals. J. Protozool.
25:235-237.
Khan, N.A., Jarroll, E.L., and Paget, T.A. 2001. Acanthamoeba can be differentiated by the.
polymerase chain reaction and simple plating assays. Curr. Microbiol. 43:204-208.
Khan, N.A. 2001. Pathogenicity, morphology, and differentiation of acanthamoeba. Curr.
Microbiol. 43:391-395.
Kilvington, S. 1989. Moist-heat disinfection of pathogenic Acanthamoeba cysts. Letters in
Appl. Microbiol. 9:187-189.
-------
Health Effects Support Document for Acanthamoeba
Kilvington, S., and Price, J. 1990. Survival of Legionellapneumophila within cysts of
Acanthamoebapolyphaga following chlorine exposure. J. Appl. Bacteriol. 68:519-525.
Kilvington, S., Larkin D.F.P., White, D.G., and Beeching, J.R. 1990. Laboratory investigation of
Acanthamoeba keratitis. J. Clin. Microbiol. 28:2722-2725.
Kilvington, S., Anthony, Y., Davies, D.J.G., and Meakin, B.J. 1991. Effect of contact lens
disinfectants against Acanthamoeba cysts. Rev. Inf. Dis. 13: (Suppl. 5)8414-415.
King, C.H., Shotts, E.B. Jr., Wooley, R.E., and Porter, K.G. 1988. Survival of coliforms and
bacterial pathogens within protozoa during chlorination. Appl. Environ. Microbiol. 54:3023-
3033.
Kingston, D., and Warhurst, B.C. 1969. Isolation of ameba from air. J. Med. Microbiol. 2:27-
36.
Knoll, H.A. 1971. Microbiology and hydrophilic contact lenses. Am. J. Optom. Arch. Am. Acad.
Optom. 48:840-844.
Kong, H.H., Kim, T.H., and Chung, D.I. 2000. Purification and characterization of a secretory
serine proteinase of Acanthamoeba healyi isolated from GAE. J. Parasitol. 86:12-17.
Kyle, D.E. andNoblet, G.P. 1987. Seasonal distribution of thermotolerant free-living amoebae.
II. Lake Issaqueena. J. Protozool. 34:10-15.
Larkin, D.F., Kilvington, S., and Dart, J.K. 1992. Treatment of Acanthamoeba keratitis with
polyhexamethylene biguanide. Ophthalmology 99:185-191.
Lawande, R.V., Abraham, S.N., John, I. and Egler, L.J. 1979. Recovery of soil amebas from the
nasal passages of children during the dusty harmattan period in Zaria. Am. J. Clin. Pathol.
71:201-203.
Ledee, D.R., Hay, J., Byers T.J., Seal, D.V., and Kirkness, C.M. 1996. Acanthamoeba griffini.
Molecular characterization of new corneal pathogen. Invest. Ophthalmol. & Visual Sci. 37:544-
550.
Lengy, J., Jakovljevich, R., and Talis, B. 1971. Recovery of a hartmannelloid ameba from a
purulent ear discharge. Harefuah 80:23-24.
9-9
-------
Health Effects Support Document for Acanthamoeba
Lindquist, T.D., Sher, N.A., and Doughman, D.J. 1988. Clinical signs and medical therapy of
early Acanthamoeba keratitis.. Arch. Ophthalmol. 106:73-77.
Ly, T.M., and Muller, H.E. 1990. Ingested Listeria monocytogenes survive and multiply in
protozoa. J. Med. Microbiol. 33:51-54.
Lyons, T.B., and Kapur, R. 1977. Limax amoebae in public swimming pools of Albany,
Schenectady, and Rensselaer counties, New York: Their concentration, correlations, and
significance. Appl. Environ. Microbiol. 33:551-555.
Ma, P., Visvesvara, G.S., Martinez, A.J., Theodore, F.H., Daggett, P.-M., and Sawyer, T.K.
1990. Naegleria and Acanthamoeba infections: Review. Rev. Inf. Diseases. 12:490-513.
Mannis, M.J., Tamaru, R., Roth, A.M., Burns, M., and Thirkill, C. 1986. Acanthamoeba
sclerokeratitis. Determining diagnostic criteria. Arch. Ophthalmol. 104:1313-1317.
Marciano-Cabral, F., Puffenbarger, R., and Cabral, G.A. 2000. The increasing importance of
Acanthamoeba infections. J. Eukaryot. Microbiol. 47:29-36.
Martinez, A.J. 1977. Free-living amebic meningoencephalitides: comparative study. Neurol.
Neruocir. Psiquiatr. 18:391-401.
Martinez, A.J., Sotelo-Avila, C., Garcia-Tamayo, J., Moron, J.T., Willaert, E., and Stamm, W.P.
1977. Meningoencephalitis due to Acanthamoeba SP. Pathogenesis and clinico-pathological
study. Acta Neuropathol. 37:183-191.
Martinez, A.J. Is Acanthamoeba encephalitis an opportunistic infection? Neurology 30:567-574.
Martinez, A.J. 1982. Acanthamoebiasis and immunosuppression. Case Report. J. Neuropathol.
Exp. Neurology. 41:548-557.
Martinez, A.J. 1985. Free-living Amebas: Natural History, Prevention, Diagnosis, Pathology,
and Treatment of Disease. CRC Press, Inc. Boca Raton, FL 33431, 156 pp.
Martinez, A.J., and Janitschke, K. 1985. Acanthamoeba, an opportunistic microorganism: A
review. Infection. 13:251-256.
Martinez, A.J., and Visvesvara, G.S. 1991. Laboratory diagnosis of pathogenic free-living
amoebas: Naegleria, Acanthamoeba, and Leptomyxid. Clin. Lab. Med. 11:861-872.
9-10
-------
Health Effects Support Document for Acanthamoeba
Martinez, A.J. 1993. Free-living amebas: Infection of the central nervous system. Mt. Sinai J.
Med. 60:271-278.
Martinez, A.J., and Visvesvara, G.S. 1997. Free-living, amphizoic and opportunistic amebas.
Brain Pathol. 7:583-598.
Martinez, A.J., Visvesvara, G.S., and Chandler, F.W. 1997. Free-living amebic infections.
pp.1163-1176 In: Pathology of Infectious Diseases, Vol. II, Connor, D.H., Chandler, F.C.,
Schwartz, D.A., Manz, H.J., Lack, E.E. (eds.) Appleton & Lange Publ.
Martinez, A.J., and Visvesvara, G.S. 2001. Balamuthia mandrillis infection. J. Med. Microbiol.
50:205-207.
Mathers, W.D., Sutphin, J.E., Folberg, R., Meier, P.A., Wenzel, R.P., and Elgin, R.G. 1996.
Outbreak of keratitis presumed to be caused by Acanthamoeba. Am. J. Ophthalmol. 121:129-
142.
McConnell, E.E., Garner, P.M. and Kirk, J.H. 1968. Hartmannellosis in a bull. Pathol. Vet. 5:1-
6.
McCulley, J.P., Alizadeh, H., and Niederkorn, J.Y. 1995. Acanthamoeba keratitis. CLAO J
21:73-76.
Mehta, A.P., and Guirges, S.Y. 1979. Acute amoebic dysentery due to free-living amoebae
treated with metronidazole. J. Trop. Med. Hyg. 82:134-136.
Meier, P.A., Mathers, W.D., Sutphin, J.E., Folberg, R., Hwang, T., and Wenzel, R.P. 1998. An
Epidemic of Presumed Acanthamoeba Keratitis That Followed Regional Flooding. Results of a
Case-Control Investigation. Arch. Ophthalmol. 116:1090-1094.
Michel, R., Miiller, K.-D., Amann, R., and Schmid, E.N. 1998. Legionella-like slender rods
multiplying within a strain of Acanthamoeba sp. isolated from drinking water. Parasitol. Res.
60:84-88.
MMWR. 1987. Acanthamoeba keratitis in soft-contact-lens wearers. Centers for Disease
Control, Atlanta, Georgia. 36:397-404.
Moore, M.B., and McCulley, J.P. 1989. Acanthamoeba keratitis associated with contact lenses:
six consecutive cases of successful management. Br. J. Opthalmol. 73:272-275.
9-11
-------
Health Effects Support Document for Acanthamoeba
Moore, M.B., McCulley, J.P., Luckenbach, M., Gelender, H., Newton, C., McDonald, M.B., and
Visvesvara, G.S. 1985. Acanthamoeba keratitis associated with soft contact lenses. Am. J.
Ophthalmol. 100:396-403.
Moura, H., Wallace, S., and Visvesvara, G.S. 1992. Acanthamoeba healyi, n.sp. and isosenzyme
and immunoblot profiles of Acanthamoeba spp., Groups II and in. J. Protozool. 39:573-583.
Munson, D.M. 1993. The distribution of cyst-forming amoebae from inshore and freshwater
Bermuda sediments. Trans. Am. Microscop. Soc. 112:88.
Nagington, J., Watson, P.G., Playfair, T.J., McGill, J., Jones, B.R., and Steele, A.D.McG. 1974.
Amoebic infection of the eye. Lancet. 2:1537-1540.
Nerad, T. 1993. Catalogue of Protists 18th ed., American Type Culture Collection, Manassas,
VA.
O'Dell, W.D. 1979. Isolation, enumeration and identification of amebae from a Nebraska lake. J.
Protozool. 26:265-269.
Ofori-Kwakye S.K., Sidebottom, D.G., Fischer, H.J., Visvesvara, G.S. 1986. Granulomatous
brain tumor caused by Acanthamoeba: a case report. J. Neuosurg. 1986, 64:505-509.
O'Malley, M.L., Lear, D.W., Adams, W.N., Gaines, J., Sawyer, T.K., and Lewis, E.J. 1982.
Microbial contamination of continental shelf sediments by wastewater. J. Water Pollut. Cntrl.
Fed. 54:1311-1317.
Ormerod, L.D., and Smith, R.E. 1986. Contact lens-associated microbial keratitis. Arch.
Ophthalmol. 104:79-83.
Page, F.C. 1967. Re-definition of the germs Acanthamoeba with description of three species. J.
Protozool. 14:709-724.
Page, F.C. 1976. An Ilustrated Key to Freshwater and Soil Amoebae. Freshwater Biological
Association. The Ferry House, Ambleside, Cumbria LA220LP, England.
Page, F.C. 1988. A New Key to Freshwater and Soil Gymnamoebae with Instructions for
Culture. Freshwater Biologicalk Association. The Ferry House, Ambleside, Cumbria LA220LP,
England.
9-12
-------
Health Effects Support Document for Acanthamoeba
Park, C.H., lyengar, V., Hefter, L., Pestaner, J.P., and Vandel, N.M. 1994. Cutaneous
Acanthamoeba infection associated with acquired immunodeficiency syndrome. Lab. Med.
25:386-388.
Penas-Ares, M., Pariagua-Crespo, E., Madrinan-Choren, R., Marti-Mallen, M., Arias-Fernandez,
M.C. 1994. Isolation of free-living pathogenic amoebae from thermal spas in N.W. Spain.
Water, Air and Soil Pollution. 78:83-90.
Pussard, M., and Pons, R. 1977. Morphologic de la paroi kystique et taxonomie du genre
Acanthamoeba (Protozoa, Amoebida). Protistologica. 13:557-598.
Radford, C.F., Lehmann, O.J., and Dart, J.K.G. 1998. Acanthamoeba keratitis: multicentre
survey in England 1992-6. Br. J. Ophthalmol. 82:1387-1392.
Regli, S., Rose, J.B., Haas, C.N., andGerba, C.P. 1991. Modeling the risk from Giardia and
viruses in drinking water. J. Amer. Water Works Assoc. 83:76-84.
Rivera, F., Ortega, A., Lopez-Ochotorena, E. and Paz, M.E. 1979. A quantitative morphological
and ecological study of protozoa polluting tap water in Mexico City, Trans. Amer. Microscop.
Soc. 98:465-469.
Rivera, F., Ramirez, P. Vilaclara, G., Robles, E., andMedina, F. 1983. A survey of pathogenic
and free-living amoebae inhabiting swimming pool water in Mexico City. Environ. Res. 32:205-
211.
Rivera, F., Galvan, M., Robles, E., Leal, P., Gonzalez, L., and Lacy, A.M. 1981. Bottled mineral
waters polluted by protozoa in Mexico. J. Protozool. 28:54-56.
Rivera, F., Roy-Ocotla, G., Rosas, L, Ramirez, E., Bonilla, P., and Lares, F. 1987. Amoebae
isolated from the atmosphere of Mexico City and environs. Environ. Res. 42:149-154.
Rivera, F., Lares, F., Ramirez, E., Bonilla, P., Rodriquez, S., Labastida, A., Ortiz, R., and
Hernandez, D. 1991. Pathogenic Acanthamoeba isolated during an atmospheric survey in
Mexico City. Microbiology of Acanthamoeba: Extended Abstracts. Rev. Inf. Dis. 13(Suppl.
5):S388-389.
Rodriguez-Zaragoza, S. 1994. Ecology of Free-Living Amoebae. Crit. Rev. Micro. 20:225-241.
9-13
-------
Health Effects Support Document for Acanthamoeba
Rohr, U., Weber, S., Michel, R., Selenka, F., and Wilhelm, M. 1998. Comparison of free-living
amoebae in hot water systems of hospitals with isolates from moist sanitary areas by identifying
genera and determining temperature tolerance. Appl. Environ. Microbiol. 64:1822-1824.
Rowan-Kelly, B., and Ferrante, A. 1984. Immunization with killed Acanthamoeba cuJbertsoni -
antigen and amoeba culture supernatant antigen in experimental Acanthamoeba
meningoencephalitis. Trans. R. Soc. Trop. Med. Hyg. 78:179-182.
Rowbotham, T.J..1980. Preliminary report on the pathogenicity ofLegionellapneumophila for
freshwater and soil amoebae. J. Clin. Pathol. 33:1179-1183.
Rude, R.A., Jackson, G.J., Bier, J.W., Sawyer, T.K., and Risty, N.G. 1984. Survey of fresh
vegetables for nematodes, amoebae, and Salmonella. J. Assoc. Off. Anal. Chem. 67:613-615.
Rutherford, I., Katanik, M.T., and Meisler, D.M. 1991. Efficacy of a chlorhexadine tablet system
for disinfection of soft contact lenses against Acanthamoeba species. Rev. Inf. Diseases.
13(suppl. 5):S416-417.
Sawyer, T.K. 1971. Acanthamoeba griffini, anew species of marine amoeba. J. Protozool.
18:650-654.
Sawyer, T.K. 1992. Distribution of microbial agents in marine ecosystems as a consequence of
sewage-disposal practices, pp. 239-262. In: A. Rosenfield & R. Mann (eds.), Disposal of Living
Organisms into Aquatic Ecosystems. Maryland Sea Grant College, Univ. MD Systems, College
Park, MD.
Sawyer, T.K., Visvesvara, G.S., and Harke, B.A. 1977. Pathogenic amoebas from brackish and
ocean sediments, with a description of Acanthameoba hatchetti, n.sp. Science. 196:1324-1325.
Sawyer, T.K., Nerad, T.A., and G.S. Visvesvara. \992.Acanthamoebajacobsi sp. n.
(Protozoa:Acanthamoebidae) from sewage contaminated ocean sediments. Proc. Hehninthol.
Soc. Wash. 59:223-226.
Sawyer, T.K., Lewis, E.J., Galassa, M, Lear, D.W., O'Malley, M.L., Adams, W.N., and Gaines,
J. 1982. Pathogenic amoebae in ocean sediments near wastewater sludge disposal sites. J. Water
Pollut. Cntrl. Fed. 54:1318-1323.
Schaumberg, D.A., Snow, K.K., and Dana, M.R. 1998. The epidemic of Acanthamoeba keratitis:
where do we stand? Cornea 17:3-10.
9-14
-------
Health Effects Support Document for Acanthamoeba
Schmoller, H. 1964. Beschreibung einiger Kulturamoeben mariner Herkunft. J. Protozool.
11:497-502.
Schuster, F.L., and Jacob, L.S. 1992. Effects of magainins on amebaand cyst stages of
Acanthamoeba polyphaga. Antimicrob. Agents Cehmother. 36:1263-1271.
Schuster, F.L. and Visvesvara, G.S. 1998. Efficacy of novel antimicrobials against clinical
isolates of opportunistic amebas. J. Euk. Microbiol. 45:612-618.
Seal, D., Stapleton, F., and Dart, J. 1992. Possible environmental sources of Acanthamoeba spp.
in contact lens wearers. Br. J. Ophthalmol. 76:424-427.
Seal, D., Hay, J., Kirkness, C., Morrell, A., Booth, A., Tullo, A., Ridgway, A., and Armstrong,
M. 1996. Successful medical therapy of Acanthamoeba keratitis with topical chlorhexidine and
propamidine. Eye. 10:413-421.
Seal, D. V. 2000. Contact-lens-associated microbial keratitis in the Netherlands and Scotland.
The Lancet 355:143-144.
el Sibae, M.M. 1993. Detection of free-living amoeba (Acanthamoeba polyphaga) in the air
conditioning systems. J. Egypt. Soc. Parasitol. 23:687-690.
Silvany, R.E., Dougherty, J.M., McCulley, J.P., Wood, T.S., Bowman, R.W. and Moore, M. B.
1990. The effect of currently available contact lens disinfection systems on Acanthamoeba
castellanii and Acanthamoeba polyphaga. Ophthalmol. 97:286-290.
Simitzis-LeFlohic, A.M., and Chastel, C. 1982. Le petits mammiferes sauvages: vecteurs
d'amibes libres? Med. Tropicale. 42:275-279.
Singh, B.N. 1952. Nuclear division in nine species of small free-living amoebae and its bearing
on the classification of the order Amoebida. Phil. Trans. Royal Soc. London. 236:405-460.
Singh, B.N., and Das, S.R. 1970. Studies on pathogenic and non-pathogenic small free-living
amoebae and the bearing of nuclear division on the classification of the order Amoebida. Phil.
Trans. Royal Soc. London, B. Biol. Sci. 259:435-476.
Singh, B.N., and Das, S.R. 1972. Occurrence of pathogenic Naegleria aerobia, Hartmannella
culbertsoni and H. rhysodes in sewage sludge samples of Lucknow. Curr. Sci., 41:277-281.
9-15
-------
Health Effects Support Document for Acanthamoeba
Singh, U. and W.A. Petri. 2000. Free-living amebas. pp. 2811-2817. In: G.L. Mandell, J.E.
Bennett and R. Dolin (eds.), Principle and Practice of Infectious Disease, 5th Ed., Churchill
Livingstone, Philadelphia, PA.
Stehr-Greene, J.K., Bailey, T.M., Brandt, F.H., Carr, J.H., Bond, W.W., and Visvesvara, G.S.
1987. Acanthamoeba keratitis in soft contact lens wearers, a case-control study. J.A.M.A.
258:57-60.
Stothard, D.R., Schroeder-Diedrich, J.M., Awwad, M.H., Gast, R.J., Ledee, D.R., Rodriguez -
Zaragoza, S., Dean, C.L., Fuerst, P.A., and Byers, T.J. 1998. The evolutionary history of the
genus Acanthamoeba and the identification of eight new 18S rRNA gene sequence types. J.
Eukaryot. Microbiol. 45:45-54.
Szenasi, Z., Endo, T., Yagita, K., and Nagy, E. 1998. Isolation, identification and increasing
importance of Tree-living' amoebae causing human disease. J. Med. Microbiol. 47:5-16.
Taylor, P.W. 1977. Isolation and experimental infection of free-living amebae in freshwater
fishes. J. Parasitol, 63:232-237.
Teknos, T.N., Poulin, M.D. Laruentano, A.M., and Li, K.K. 2000. Acanthamoeba
rhinosinusitis: characterization, diagnosis, and treatment. Am. J. Rhinol. 14:387-391.
Telang, G.H., Scola, F., Kantor, G.R., Stieritz, D., and Reboli, A.C. 1996. Disseminated
Acanthamoeba infection in a patient with AIDS. Inf. Dis. Clin. Practice. 5:387-389.
Thamprasert, K., Khunamornpong, S., and Morakote, N. 1993. Acanthamoeba infection of
peptic ulcer. Ann. Trop. Med. Parasitol. 87: 403-405.
Theodore, F.H., Jakobiec, F.A., Juechter, K.B., Ma, P., Troutman, R.C., Pang, P.M., and
Iwamoto, T. 1985. The diagnostic value of a ring infiltrate in Acanthamoeba keratitis.
Ophthalmol. 92:1471-1479.
Tyndall, R.L., Lyle, M.M., and Ironside, K.S. 1987. The presence of free-living amoebae in
portable and stationary eye wash stations. Am. Ind. Hyg. Assoc. J. 48:933-934.
United States Food and Drug Administration. 1973. Microbiological guidelines for new contact
lenses. U.S. Food and Drug Administration, Washington, D.C.
9-16
-------
Health Effects Support Document for Acanthamoeba
van Klink, F., Alizadeh, H., Stewart, G.L., Pidherney, M.S., Silvany, R.E., YuGuang, H.,
McCulley, J.P., and Niederkorn, J. Y. 1992. Characterization and pathogenic potential of a soil
isolate and an ocular isolate of Acanthamoeba castellanii in relation to Acanthamoeba keratitis.
Curr. Eye Res. 11:1207-1220.
Visvesvara, G.S. 2001. Personal communication.
Visvesvara, G.S., Jones, D.B., and Robinson, N.M. 1975. Isolation, identification, and biological
characterization of Acanthamoeba polyphaga from a human eye. Am. J. Trop. Med. Hyg.
24:784-790.
Visvesvara, G.S. 1987. Laboratory Diagnosis. In. Rondanelli, E.G., ed. Amphizoic amoebae
human pathology. Padua, Italy Piccin Nuova Libraria, 193-215.
Visvesvara, G.S. 1995. Pathogenic and opportunistic free-living amoebae. In: Manual of Clinical
Microbiology, 6th ed.; P.R. Murray, E.J. Baron, M.A. Pfaller, EC. Tenover, and R.H. Yolken,
eds. pp. 1196-1203. ASM Press, Washington, D.C.
Visvesvara, G.S., Neafie, R.C., and Martinez, A.J. 1997. Pathogenic and opportunistic free-
living amoebae. In: Pathology of Emerging Infections. Horsburgh, C.R. Jr. and Nelson, A.M.
(eds.). Am. Soc. Microbiol, Washington, D.C., pp. 257-267.
Visvesvara, G.S., Mirra, S.S., Brandt, F.H., Moss, D.M., Mathews, HJVL, and Martinez, A.J.
1983. Isolation of two strains of Acanthamoeba castellanii from human tissue and their
pathogenicity and isoenzyme profiles. J. Clin. Microbiol. 18:1405-1412.
Volker-Dieben, H.J.M., Bos, H.J., Kok-van Alphen, C.C. 1980. Amebic keratitis in a wind-
surfer. Ned. Tijdschr. Geneeskd. 124:1147-1151.
Walker, P.L. Prociv, P., Gardiner, W.G., and Moorehead, D.E. 1986. Isolation of free-living
amoebae from air samples and an air conditioner filter in Brisbane. Med. J. Australia. 145:175.
Walochnik, J., Obwaller, A., and Aspock, H. 2000. Correlations between morphological,
molecular biological, and physiological characteristics in clinical and nonclinical isolates of
Acanthamoeba spp. Appl. Environ. Microbiol. 66:4408-4413.
Walochnik, J., Haller-Schober, E., Kolli, H., Picher, D., Obwaller, A., and Aspock, H. 2000.
Discrimination between clinically relevant and nonrelevant Acanthamoeba strains isolated from
contact lens-wearing keratitis patients in Austria. J. Clin. Microbiol. 38:3932-3936.
9-17
-------
Health Effects Support Document for Acanthamoeba
Wiley, C.A., Safrin, R.E., Davis, C.E., Lampert, P.W., Braude, A.I, Martinez, A.J., and
Visvesvara, G.S. 1987. Acanthamoeba meningoencephalitis in a patient with AIDS. J. Inf. Dis.
155:130-133.
Wilhelmus, K.R., and Jones, D.B. 1991. Program planning for research on Acanthamoeba. Rev.
Infect. Dis. Suppl. 5:8446-450.
Wilson, D.E., Bovee, B.C., Bovee, G.J., and Telford, S.R.. Jr. 1967. Induction of amebiasis in
tissues of white mice and rats by subcutaneous inoculation of small free-living, inquilinic, and
parasitic amebas with associated coliform bacteria. Exper. Parasitol. 21:277-286.
Wright, P., Warhurst, D., and Jones, B.R. 1985. Acanthamoeba keratitis successfully treated
medically. Br. J. Ophthalmol. 69:778-782.
Yammaura, H., Shirasaka, R., Matsumoto, K., Nakai, T., Horikami, H., and Ishii K. 1993.
Acanthamoeba in house dust. Jpn. J. Parasitol. 42:130-133.
Yamaura, H., Shirasaka, R., Matsumoto, K., Kuwabara, M., Tsuji, M., and Horikami, H. 1993.
Isolation of Acanthamoeba from sandboxes in Tokyo and Hiroshima City. Jpn. J. Parasitol.
42:361-364.
Yang, Y.F., Matheson, M., Dart, J.K., and Cree, LA. 2001. Persistence of acanthamoeba antigen
following acanthamoeba keratitis. Br. J. Ophthalmol. 85:277-280.
Zagardo, M.T., Castellani, R.J., Zoarski, G.H., and Bauserman, S.C. 1997. Granulomatous
amebic encephalitis caused by leptomyxid amebae in an HIV infected patient. Am. J.
Neuroradiology 18:903-908.
9-18
------- |