United States
 Environmental Protection
 Agency
Office of Science
and Technology
Washington, D.C.
EPA/822/R/01/003
December 31,2001
»  EPA  Office of Water
  Health Effects Support Document for Acanthamoeba
    PROPER LENS
    CARE CAN
    PREVENT
    INFECTION
             EXTERNAL REVIEW REPORT

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 Health Effects Support Document                               EPA/822/R/01/003

                             ACKNOWLEDGMENTS

                                     Authors
                              Charles P. Gerba, Ph.D.
                               University of Arizona

                              Nena Nwachuku, Ph.D.
                           Office of Science & Technology
                                     USEPA

 External Expert Reviewers;
 Govinda Visvesvarja, Ph.D. Centers For Disease Control, Atlanta, GA.
                 i
 A. Julio Martinez, M.D.    Dept. of Neuropathology, University of Pittsburgh, PA.

 Walter Jakubowski        WaltJay Consulting

 Jerry Niederkorn, Ph.D.    Dept. of Ophthalmology, University of Texas Medical Center

 Hassan Alizadeh, Ph.D.   Dept. of Ophthalmology, University of Texas Medical Center

 Hercules Moura, Ph.D.    Centers For Disease Control, Atlanta, GA.
                 !
 Internal EPA Peer Reviewers
 Rita Schoeny, Ph.D.       U.S. EPA, Office of Science and Technology
Paul S. Berger, Ph.D.

Guy Carruthers

David Soderberg

Al Dufour, Ph.D.

James Sinclair, Ph.D.

Management Support
Geoffrey Grubbs

Jeanette Wiltse, Ph.D.
U.S. EPA, Office of Ground Water Drinking Water

U.S. EPA, Office of Ground Water Drinking Water

U.S. EPA, Office of Ground Water Drinking Water

U.S. EPA, Office of Research and Development, Ohio

U.S. EPA, Office of Research and Development, Ohio


Director, Office of Science and Technology, U.S. EPA

Director, Health and Ecological Criteria Division
                 i
      Support provided under EPA contract #68-C-99-232

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                          Health Effects Support Document
                             TABLE OF CONTENTS
               ' !
LIST OF TABLES	,.	iii

LIST OF FIGURES	.	  iv

GLOSSARY OF TERMS	v

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	 3-3
      3.3 Methods; of Identification			..3-6
      3.4 Cultivation	 3-6
      3.5 Significance of Endosymbiosis	 3-7

4.0 OCCURRENCE	 4-1
      4.1 Water .l.	 4-2
            4.1.1  Surface Waters	 4-2
                  4.1.1.1 Freshwaters	4-2
                  4.1.1.2 Seawater		4-3
            4.1.2;  Tapwater and Bottled Water		4-3
            4.1.31  Swimming Pools and Spas	4-5
            4.1.4|  Sewage and Biosolids		4-6
      4.2 Animal Wastes	4-7
      4.3 Air, Dust and Soil	;	4-7
      4.4 Summary	'.	4-8

5.0 HEALTHEFFECTS	 5-1
      5.1 Eye Infections (Acanthamoebic Keratitis)	5-3
             5.1.1   Symptoms ofAcanthamoeba Keratitis	 5-7
             5.1.2   Diagnosis ofAcanthamoeba Keratitis	 5-8
             5.1.31   Identification Procedures	 5-9
             5.1.4   Treatment of Acanthamoebic Keratitis	^	5-10
             5.1.51   Incidence ofAcanthamoeba Keratitis	5-12
             5.1.6   Pathogenicity	5-13
             5.1.7   Immunity	5-15
      5.2 Granulomatous Amoebic Encephalitis	5-16
             5.2.1   Diagnosis and Treatment of GAE	5-19
             5.2.2'   Incidence of GAE	  5-20
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             5.2.3   Pathogenesis and Immunity	  5.21
       5.3  GAE in Domestic Animals and Wildlife	  5.21
       5.4  Other Infections caused by Acanthamoeba	.., 5-22
       5.5  Immunocompromised Individuals	  5.22
       5.6  Children	!			..  5.24
       5.7  Effect of Endosymbiosis on Virulence	   5-24
                                       i
6.0 RISK ASSESSMENT	\	6-l
      6.1  The Organism and its Occurrence (Exposure)	6-1
      6.2  Epidemiological Evidence for Acanthamoeba Keratitis Transmission by Tap water6-1
      6.3  Resistance to Drinking Water Treatment and Disinfection	 6-3
      6.4  Dose Response	i	6-5
      6.5  Risk Characterization	.	6-7

7.0 PREVENTION OF ACANTHAMOEBIC KERATITIS ASSOCIATED WITH CONTACT
      LENSES	.....!...	.7-1
      7.1  Types of Contact Lenses	;	7.1
      7.2  Demographics .of Contact Lens Usjs	7-2
      7.3  Risk Factors  	i	7.4
      7.4  Contact Lens Disinfection	\	7.5
            7.4.1 Studies of Lens Disinfection	7-6
            7.4.2 Hydrogen Peroxide .....:..	7-8
            7.4.3 Multi-Purpose Solutions . J	 7.9

8.0 DATA GAPS	8-1
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9.0 REFERENCES	9.1
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                                 LIST OF TABLES


Table 2.1 Major Waterborne/Water-based Pathogenic Protozoa	 2-2


Table 3.1 Currently Identified Species of Acanthamoeba	3-2
                  i

Table 3.2 Acanthamoeba Species Classification ...	 3-3


Table 3.3 Bacterial Endosymbionts of Acanthamoeba	3-9


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)	5-3


Table 5.2 Characteristics and Symptoms of Patients with Acanthamoeba Keratitis	5-4


Table 5.3 Incidence of Acanthamoeba Keratitis	5-13


Table 6.1 Human Infection Caused by Species of Acanthamoeba 	6-3


Table 6.2 Mechanisms involved hi Acanthamoeba Keratitis	6-8


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 hi 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 	7-5


Table 7.7 Risk Factors for Acanthamoebic Keratitis in Contact Lens Wearers	7-5
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                                LIST OF FIGURES


Figure 3.1 Life Cycle ofAcanthamoeba Species	3-4

Figure 3.2 Acanthamoeba trophozoite. Note the characteristic spine-like acanthopodia	3-5

Figure 3.3 Cysts ofAcanthamoeba. Note the characteristic double wall with an outer wrinkled
                ectocyst and an inner polygonal endocyst	3-6

Figure 3.4 Significance of Endosymbiosis to Waterborne Disease Transmission .......... 3-10

Figure 5.1 Life Cycle ofAcanthamoeba spp. and Human Infection	5-2

Figure 5.2 Slit lamp view showing a paracentral complete ring infiltrate of the cornea	 5-8

Figure 5.3 Normal Eye	j	,	5-8

Figure 5.4 Granulomatous Amoebic Encephalitis (GAE) 	5-18

Figure 6.1 Eye Trauma and Contact Lenses as Determinants of Susceptibility to
          Acanthamoeba Keratitis	'	6-6
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Amphizoic amoeba

Anterior uveitis

Axeriic

Cytopathogenic effects


Confocal microscopy

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Cornea

Endocyst


Endosymbiosis


Epithelium


Exocyst

Free-living

Granulomatous
amoebic encephalitis
Hematogenous spread

Keratitis

IgA

IgG
     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

 Alteration of the appearanc of animal cells hi culture due to the
 growth of pathogenic microorganisms

 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

 Spread through the blood

 Inflammation of the cornea

 The predominant antibody class present in secretions

 The predominant antibody present in human serum
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Macrophage


Meningoencephalitis

Nodular scleritis

Ocular

Phagocytosis

Ring infiltrate



Sclera


Scleritis

Stroraa

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
                i                            •
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 anld chronic

The second vascular coat of the eye, lying immediately beneath the
sclera. It consists jof iris, ciliary body, and choroid.
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                            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


 microbiologists1 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
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 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
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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
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of then* ability to live both free hi 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
                                           i
Escherichia coliorKlebsiella pneumoniae.    '



The genus Acanthamoeba consists of as many as 20 species classified hi three groups based on

cyst morphology. Several species of Acanthamoeba are known to cause infections in humans.

They include A. astronyxis, A. castellanii, A. culbertsoni, A. divionensis, A. griffini, A. healyi, A.

rhysodes, A. hatchetti, A. palestinensis and A. polyphaga. Contaminated recreational and tap
       '                                    j
water have been implicated as sources of exposure, especially for those species causing infections
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of the eye. No studies are available on Acanthamoeba spp. in drinking water hi 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.
Two types of illnesses are most commonly associated with Acanthamoeba. These are
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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 confpcal microscopy, diagnostic tests usually rely on demonstrating amoebae on
                  i    '  .
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 becominjg 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
                  i
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 hi the

United States, and 71 million people globally wear contact lens. Every individual who wears
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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
                                           I                •                       "i,
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-polyrnixin B
                                           i
sulfate-gramicidin,  oral itraconazole, topical micionazole, polyhexamethylene biguanide (PHMB),
                                           i                                          ' i
                                           i
and topical clotrimazole.                     \
Options for lens disinfection include chlorohexidine, benzalkonium chloride, and hydrogen

peroxide. Of these, hydrogen peroxide is the moist effective chemical disinfectant against bacteria

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
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without the need for neutralization of the disinfectant before lens use. Multi-purpose solutions


provide the easiest technique for the lens wearer to clean and disinfect the lens and better


compliance results have been demonstrated. Multi-purpose solutions contain a detergent with a


polyquaternium or polyhexamethylene biguanide (PHMB), hi 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 hi 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.

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Worldwide, the incidence of microbial keratitis has been reported to range from 1.1 to 2,000/106


among contact lens wearers. Difficulties  in the diagnosis of Acanthamoeba keratitis probably

leads to an underestimation of the true number of cases.
Molecular-based investigations have established domestic tapwater as a proven source of


Acanthamoeba infection in lens wearers. The organisms have been isolated from household taps


and probably feed on the microbial biofilm within the distribution system. An epidemiological


study in the midwestern United States suggested that an epidemic of presumed Acanthamoeba


infection was associated with municipal water supplies subjected to flooding during 1993-1994.


The incidence of Acanthamoeba was ten times greater (1.30 vs. 14.3 cases/106) in areas affected


by flooding. The incidence was also significantly lower if the home was supplied with tapwater
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from a private well.  Studies suggest that the risk of Acanthamoeba keratitis may be related to

concentrations of the organism present in surface waters and tapwater.
Granulomatous amoebic encephalitis (GAE) caused by Acanthamoeba is the second major


infection associated with Acanthamoeba. GAE is now recognized as a disease occurring most


often in people with poor immune systems or other debilitating health problems. Predisposing

factors include chemotherapy, dialysis, diabetes, treatment with steroids, smoking, or acquired

immunodeficiency syndrome. The symptoms of GAE during the initial stage of the disease are

indistinguishable from bacterial and viral meningitis.  The amoeba is believed to enter the

bloodstream, probably via the nose, lungs, or breaks in the skin following injury or trauma.

Successful treatment is rare. Pentamidine, propamidine, miconazole, ketoconazole, sulfadiazine,

itraconazole, fluconazole, and 5-fluorcytosine may be effective in treating GAE, and efforts to

find at least a partially successful treatment are in progress.
The global incidence of recorded GAE cases due to Acanthamoeba was 120 cases as of the year


2000, 84 of those occurred in the U.S. and over 50 of the GAE cases were found in AIDS patients.
             t                             i

An estimate ofAcanthamoeba keratitis cases in j the U.S. stood at 500 with over 3000 cases


worldwide. There is general agreement that both GAE and keratitis have significantly increased


in the last 10 years in the U.S. because of the increase in the use of contact lens wearers of all

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ages for various reasons including athletic and cosmetic reasons, and the increase in the number


of immuno-suppressed individuals.           i
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 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

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 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 at 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
                   t

 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 heterotrpphic bacteria.
 While it is clear that; a relationship exists between Acanthamoeba in water and keratitis, the role
                   I

 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
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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

                                           i

supplies, was 14/106. Even if all the cases ofAcanthamoeba 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
                                           i


water supplies.                             :

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The risk of keratitis is clearly greater for contact lens wearers. If consumers follow contact lens


manufacturers'instructions and lens care produc^: instructions for storage and rinsing of lenses,
                                           i

keratitis would be greatly reduced. Proper contabt lens care and disinfection are essential for



preventing infection by Acanthamoeba.        ;
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A significant data gap is the absence of information on the occurrence ofAcanthamoeba spp. in
                                           i

tapwater in the United States.  Information on the concentration ofAcanthamoeba 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.
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                                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, Cryptosporidiwn


spp., Naegleriafowleri and certain Acanthamoeba spp (Table 2.1).


                  [


Acanthamoeba are free-living amoebae which have no defined shape. They move by pseudopods,

                  I
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 Sas 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 arid as pathogens hi 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


then- morphology (Table 3.2). Unlike Naegleriafowleri, the most important species ofNaegleria
                  r '                 .                                 •

that causes human disease, several species of Acanthamoeba are known to cause infections in


humans. They include A. astronyxis, A. castellanii, A. culbertsoni, A. divionensis, A. healyi, A.


rhysodes, A. hatchetti, A. palestinensis and A. polyphaga. Exposure to contaminated recreational
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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 abcess)

diarrhea

fever, loss of fetus
diarrhea
diarrhea
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               3.0 GENERAL INFORMATION AND PROPERTIES
                  !
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3.1 History and Taxonomy
                  r
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 0/.(1958) found that it could cause meningoencephalitis in experimentally infected

animals. Results of studies with laboratory animals led to the finding that these free-living

amoebae had caused fatal meningitis in several patients. The term "free-living pathogenic

amoebae", or PFLA; has been used to describe these opportunistic pathogens. They are now

referred to as amphizoic amoeba (Page, 1967).



Taxonomy of Acanthamoeba is a contentious area. Those species now known as Acanthamoeba
                  i
were previously placed in the genus Hartmanella, but in 1967 they were definitely classified as a
                  i
separate genus by Page (1967). Pussard and Pqns (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
                  i
(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
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human pathogens belong to Group II, although^, culbertsoni, from Group III, is also a

recognized pathogen.

               Table 3.1 Currently Identified Species of Acanthamoeba
                        Species
 Species
                       A. astronyxis
                       A. castellanii
                       A. comandoni
                       A. culbertsoni
                       A. divionensis
                       A. echinulata
                       A. gigantea
                       A. grifflni
                       A. hatchetti
                       A. healyi
                       A.jacobsi
                       A. lenticulata
                       A. lusdunensis
 A. mauritaniensis
 A. palestinensis
! A. paradivionensis
\ A. pearcei
\ A. polyphaga
 A. quina
'' A. rhysodes
\ A. royreba
 A. stevensoni
\ A. terricola
\ A. triangularis
 A. tubiashi
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      Table 3.2 Acanthamoeba Species Classification (Pussard and Pons, 1977)
                  Group I
Group II
Group III
                  A. astronyxis   A.castellani        A. palastinensis
                  'A. comandoni   A. mauritaniensis    A. culbertsoni
                  A. echinulata   A. polyphaga
                  :               A. lugdunesis
                                 A. quina
                                 A. rhysodes
                                 A. divionensis
                  '•               A. paradivionensis
                                 A. griffini
                  	A. triangularis
                   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 pm 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 pm in length) (Figure 3.3).

It has one nucleus and is double-walled, with a wrinkled proteinaceous outer ectocyst and an
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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).

                                        I


                   Figure 3.1 Life Cycle of Acanthamoeba species
                            Vegetative form or trophozoite
                                   Reproduction by
                                  binary fission with
                                 dissolution of nuclear
                                membrane at prophase
                                     Encystment
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 Figure 3.2 Acanthamoeba trophozoite (amebic stage). Note the characteristic spine-
                       like acanthapodia. (Visvesvara, 1987)
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.

                 i


Acanthamoeba are carriers of intracellular bacteria, especially Legionella species, which have the

ability to reproduce within the trophozoite. It has been proposed that this may be of importance

in the persistence and spread of these organisms in the environment (King et al, 1988).
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   Figure 3.3 Cysts of Acanthamoeba. Note the characteristic double wall with an
 outerwrinkled ectocyst and an inner polygonal endpcyst (Visvesvara, unpublished)
3.3 Methods of Identification
                                        i
The identification of individual species of Acanthamoeba is based on morphological

observations, but recent taxonomic studies have employed isoenzyme (de Jonckheere, 1S>87) or

mitochondrial DNA restriction endonuclease analysis in an attempt to form a classification
                                        i                     -                 -   '
system. A study of mitochondrial DNA has produced comparable results. In the first study, 33
                                        !
strains, of which 30 were corneal isolates, were separated into ten groups according to restriction

length pattern polymorphism.               ,
3.4 Cultivation

Acanthamoeba are easily grown on non-nutrient agar plates seeded with Escherichia coli or

Klebsiella pneumoniae (Kilvington et al., 1990; Visvesara et al., 1975). One of the more
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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 axehic (bacteria-free) media may be found in a
publication by the American Type Culture Collection (Nerad, 1993). Since some species of
                  f
amphizoic amoeba grow at mammalian body temperatures, many labs incubate replicate
cultures at room temperature, 37°C to 45°C, or higher.
                  I  -
                  f          -          •
3.5  Significance of Endosvmbiosis
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

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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 this virulence of Legionella (Dowling et al, 1992).




It may also be involved in the observed phenomenon that L. pneumophila can be viable but non-




detectable by cultivation on agar-based systems (Connor et al., 1993). Hay and Seal (1994b)




have proposed that the latter observation may have profound implications with regard to




surveillance of water systems far 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 hi 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
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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 ayium
                           Burkholderia picketti
                           Vibrio cholerae
                           Francisetta tularensis
                           Chlamydia pneumoniae
                           Rickettsiales
                           Listeria mono'cytogenes
                           Fritsche et al.,-1999; Ly and Miller, 1990*
                          *live within the Acanthamoeba
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    Figure 3.4 Significance of Endosymbiosis to Waterborne Disease Transmission
                   Amoeba
Bacteria
                     Enhanced virulence
                      of both organisms
  Endosymbiotic
relationship develops
                Enhanced resistance
                   of bacteria to
                   disinfectants
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                               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
Rivera el al, 1979
Rivera etal, 1981
Rohr et al, 1998
Tyndall et al, 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
Sawyer et al, 1982
Yamauraefa/., 1993
Singh, 1952
Yamauraefa/., 1993
Rude et al, 1984
Taylor, 1977
Walker et al, 1986
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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 hi
                                         i •

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 hi 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
                                         I

dissolved oxygen,  attenuation, and water temperature. Information on amphizoic amoebae from


this study showed that hi 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, D.C. to tidal waters (brackish) 0.8 m below a municipal sewage
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treatment plant. They identified seven species of acanthamoeba, most of which occurred in the


tidal portion of the river near the sewage treatment plant. John and Howard (1995) processed


2,016 samples from ponds in Oklahoma and recovered 34 strains of pathogenic (induced brain


damage) amoebae with 35 percent identified as Acanthamoeba. They estimated that there was


approximately  1 pathogen per 60 samples, and 1 pathogen per 3.4 liters of water. They found the


highest percentage of pathogens during spring and fall, while Kyle and Noblet (1987) found


summer and fall to be the peak periods.





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 et al (1992) recovered several species of Acanthamoeba from sewage-


contaminated inshore New York and New Jersey shellfish beds that periodically were closed to


shellfish harvesting, i Munson (1993) recovered several species of Acanthamoeba from coastal
               -  . i

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
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occurrence, however; all of these studies have been done in countries other than the United
                                         i •     -
States. Rivera et al. (1979) collected 25 one-gallon water samples from faucets in private

residences in Mexico. Flagellates were found in 84% of the samples, amoebae in 13% and

ciliates in 1.9%. Although found infrequently, Acanthamoeba astronyxis and A. castellanii were


recovered from the same samples. In another study, Hamadto et al. (1993) tested 50 tap water

samples in Egypt and recovered unidentified species of Acanthamoeba from two  of them.

Michel et al. (1998) tested drinking water in a new hospital hi 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 hi Germany, and

found amoebae hi 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
                                         i

kitchen cold water tap supplied by the mains. When 41 strains of amoebae were recovered from
                                         i.            ...

49 swab samples collected from moist areas hi the hospital, such as walls, floor tiles, and sinks,

22 percent were species of Acanthamoeba. In a more recent study hi 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.


                                        ' i
                                         i                •                   •
The common occurrence of Acanthamoeba hi eye wash stations filled with tapwater containing

free chlorine (concentration of chlorine was not reported) has been reported hi the United States


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 (Bowman et at., 1906). 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 ndt 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.




 4.1.3 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 hi New York disinfected

 with either chlorine or bromine and recovered amoebae from 27 of them. The species were not

 identified but. were referred to as belonging to the "Hartmannella-Acanthamoeba" group, a term

 often used before the two genera were recognized as distinct taxonomic entities. Acanthamoeba

 has been in swimming pools or other bodies of water around the world, including Germany

 (Janitschke et al, 1980), Mexico (Rivera et al, 1983) and frozen swimming areas in Norway

 (Brown and Cursons, 1977).
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 Thermal bathing pools (spas) are also sources [for potentially pathogenic amoebae (Martinez,
                                         i
 1985). Brown et al. (1983) tested 9 thermal pools hi New Zealand and identified temperature
                                         i
 tolerant strains ofAcanthamoeba 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 jfrom natural springs of thermal water. They

 recovered 12 strains ofAcanthamoeba from cultures incubated at 42°C to 45°C. Two strains were

 identified as A. castellanii, one as A. lugdunensis and the others as Acanthamoeba spp. All were

 pathogenic to mice. The authors conducted a second study (Rivera et al, 1991) and recovered A

 culbertsoni and A polyphaga from heated physiotherapy tubs. Penas-Ares et al.  (1994) tested

 heated water used to fill 12 spas in Spain. The water was classified as sulphurous, and

 temperature ranged from 34°C to 64°C. The authors recovered 13 strains of amoebae from 8 of

 the spas. Four of the 8 spas yielded A. polyphdga 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
                                         i
Daggett (1982) published a description of potentially pathogenic Acanthamoeba and Naegleria

hi polluted waters with emphasis on health risks to divers. Singh and Das (1972) studied biosolid
                                         i
samples in Bombay, India and recovered strains ofAcanthamoeba culbertsoni and A. rhysodes

that were pathogenic to mice. Bose et al. (1990) extended studies on sewage hi India to include
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 Calcutta, where they isolated a pathogenic strain of A. castellanii and a non-pathogenic strain of

 A. astronyxis.




 4.2 Animal Wastes
                  i

 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
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twelve children harbored .4. rhysodes. Lawande (1979) also exposed open culture plates to the
                                          1

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. (19J87) conducted similar studies during the rainy

                                          i               . ':
season hi Mexico City, Mexico. They recovered A. astronyxis A. castellanii, A. culbertsoni, and
                                          !
                                          I
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

                                          i
microbial pathogens including Acanthamoeba in the atmosphere (Walker et al., 1986; Ma et al.,


1990; el Sibae, 1993)f Kingston and Warhurst (1969) conducted quantitative studies on the


density of Acanthamoeba cysts hi outdoor air. They recorded values of one cyst per m3 and one


cyst of A. castellanii per 18.3 m3 of air.
4.4  Summary


Acanthamoeba can be isolated from most aquatic environments, air, and soil. Their concentration


in water is related to the number of bacteria upon which they feed. Little quantitative
                                          i

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

                                          i
exposure may occur because of their presence in swimming pools, hot tubs and surface waters.


They may occur seasonally in greater numbers in the early spring and early fall. The occurrence


of Acanthamoeba in the environment is summarized hi Table 4.1.
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                              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 hi 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.            ;


                  i

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.
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         Figure 5.1 Life cycle of Acanthamoeba spp. and Human Infection
                                          INVAOC
                                  OTHER TISSUE*
                   crsr
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   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 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
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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
                                          i
                   • 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 hi 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
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dust while bailing 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 tp 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 hi 1 of 59 commercial saline

                ,  i                             '                                   '
solutions, 11 of 11 homemade solutions, and 23 of 29 bottles of non-sterile distilled water. Thus,
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 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
                                          i

, literature on amoebic eye diseases beginning vyith some of the earliest recognized cases aind


 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

                                          t
 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


daily wear (56%) or extended wear soft (19%). 'Some patients (including both contact lens
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wearers) (26%) had, a history of corneal trauma before developing acanthamoebic keratitis, and


25% of patients had a history of exposure to contaminated water.




Two studies have identified tapwater washing of lens cases in cases of Acanthamoeba (Seal et


al, 1997, Ledee et dl, 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

                  i
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.
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   Figure 5.2 Slit lamp view showing a paracentral complete ring infiltrate of the
        cornea. The ring infiltrate is diagnostic ofAcanthamoeba infections
                              (Theodore et al., 1985)
Ring infiltrate
                                                           Figure 5.3  Normal eye
5.1.2 Diagnosis ofAcanthanweba Keratitis
                                       i
While positive diagnosis of acanthamoebic keratitis can be made by in vivo confocal

microscopy, diagnostic tests usually rely on demonstrating amoebae on cornea! scrapings or

biopsy material (Seal et al., 1996). Samples of cornea! epithelium and any infiltrated stroma

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 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
                  i
 seeded with E. coli (Singh and Petri, 2000). With incubation at 32°C in air, migration tracks are

 usually visible within 48 hours. Positive identification requires some experience, and it is useful

 to incubate a control plate that is not inoculated with a clinical specimen.



 5.1.3  Identification Procedures

 Standard methods for morphological characterization, isoenzyme electrophoresis, immunological

 techniques, and temperature tolerance tests have been published and widely used (Singh and

 Petri, 2000). Results obtained by using one or more of these techniques, coupled with animal

 pathogenicity tests, and the shape and size of cysts, are often adequate for identifying more

 commonly occurring species of Acanthamoeba.
Corneal biopsy of infected eye are usually sufficient for confirming infection by amphizoic

amoebae. However, it may be possible to make an identification of genus when distinctive

double-walled wrinkled cysts suggest a Group El 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
                  I
Michel, 1988) are extremely useful in combination with morphological  features for identifying
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 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 Kerjatitis

 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.
                                         I
 In 1985, Wright et al. reported successful medical treatment using propamidme 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
                                         i •               .
 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

 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-
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 polymixin B sulfate-gramicidin; in a series reported recently a medical cure was achieved in 11


 of 14 patients with jeye 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 hi 1992 Larkin et a/.reported its successful clinical use at a concentration of
                  I

 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 hi 11 of 12 patients (Seal et al, 1996)
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 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
                                         i

 Table 5.3. The incidence of all causes of micrpbial keratitis (largely bacterial) is about 400/106
                                         i

 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).
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1
Table 53 Worldwide Incidence of Acanthamoeba Keratitis
!
Incidence per Population Country Year(s) Reference
1,000,000
1.65 to 2.01
1.1
149
0.58 to 0.71
1.40
1.30
14.3
Contact Lens USA
Wearer (CLW)
CLW Netherlands
CLW UK
General USA
Population (GP)
GP UK
GP- Iowa well USA
water
GP - during USA
flooding
municipal
systems
1985-1987 Schaumberg et al,
1996 Cheng et al, 1999
1996 Seal; 2000
1985-1987 Schaumberg et al.,
1998


1998
1 996 Radford etal.,1998
1993-1994 Meier et al. , 1998
1993-1994 Meier et al, 1998


5.1.6 Pathogenicitv

The pathogenesis of acanthamoebic keratitis has been suggested to follow two pathways
                  i
(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 hi the initiation of infection is

the attachment to the epithelial surface. Amoebae bind to the corneal surface and produce

epithelial thinning and necrosis.
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The pathogenicity ofAcanthamoeba 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

                                          i               , . .. .
                                          I


pathogenic amoeba by formation of amoebastohe (characteristic of amoeba phagocyte) arid that




Acanthamoeba phageocytosis may be both an efficient means of obtaining nutrients and a




significant factor in pathogenesis ofAcanthamoeba infections. Khan et al. (2001) differentiated




pathogenic Acanthamoeba by their ability to produce cytopathogenic effects (CPE) on comeal




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 ofAcanthamoeba 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 ofAcanthamoeba may be an individuals ability to produce
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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-
                  I
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 aati-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
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 can be due to persisting acanthamoebic antigens. Yang et al. (2001) found that Acanthamoeba



 cysts were found to persist for up to 31 months in the eye after treatment although trpphozoites



 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.








 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 hi 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




Granulomatous amoebic encephalitis (GAE) caused by Acanthamoeba spp. is the second major



infection associated with Acanthamoeba. GAE is a chronic, progressive disease of the central



nervous system occurring most often hi persons with poor immune systems or other debilitating



health problems.  Predisposing factors include chemotherapy, dialysis, diabetes mellitus,

                                          f

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
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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, Naegleriafowleri, 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
                  i

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 hi 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.
                  i

Balamuthia has been identified in approximately 40 patients hi the United States (U.S.),


including >10 with ADDS infection (Martinez et al., 1997, Visvaresvara, 2001). In c'ontrast,
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Acanthamoeba has accounted for approximately 84 (-50 with AIDS) cases in the U.S. and 120

worldwide (Martinez et al., 1997, Visvaresvara, 2001). The disease may be the end result of

long-term injury. Fatal infections probably occur in individuals with extensive damage to the

central nervous system and internal organs prior to the manifestation of overt clinical symptoms.
 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
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 esitry

through the nasal passages, lower respiratory system or breaks in the skin caused by injury (Ma

et al., 1990). Patients who have been treated for GAE range from children to elderly adults with
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a clinical history of illness ranging from about 1 week to 6 months (Martinez et al, 1977).

Symptoms of neurological disease upon admission to a hospital are varied, including headache,

drowsiness, low-grade fever and stiffness of the neck. Other symptoms that may appear early in
                     !>
the disease are personality changes, seizures, nausea, vomiting or lethargy (Martinez and
                  t
Visvesvara, 1991). ;Thorough diagnostic procedures are necessary to recognize amoebic

meningoencephalitis because upon initial examination, the disease is not always easy to

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
                  i
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 hematpma (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
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such as pentamidine, propamidine, miconazole, ketoconazole and 5-fluorocytosine may be

effective in treating GAE (Martinez et al., 1997). There are some occasions when skin nodules

harboring Acanthamoeba are detected prior to spreading to internal organs and the central

nervous system. Visvesvara et al. (1997) suggested that when skin nodules or ulcers are present,
                                                      \
treatment may be tried using topical chlorhexidine gluconate and intravenous pentamidine.
In spite of the poor prognosis for most patients with GAE, efforts to find at least a partially

successful treatment are in progress. A new class of peptide compounds called magainins that

may have amoebostatic and amoebicidal properties when used with other amoebicidal agents

(Martinez et al. 1997, Schuster and Jacob, 1992). Schuster and Visvesvara (1998) tested

antimicrobials and phenothiazine compounds against amphizoic amoebae and found the levels
                                          i                  *            '  .   .   i   -
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,

2001). 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 hi the number of imrnunosuppressed
                                          !
individuals (Marciano-Cabral et al, 2000; EPA, 1998).
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 5.2.3 Pathogenesis and Immunity




 The pathogenesis of GAE is complex and poorly understood (Martinez and Visvesvara, 1997).




 In GAE, the immunity is predominantly T-cell mediated, therefore the dimunition of CD+ and T




 helper lymphocytes, as occurs in AIDS patients, enables the proliferation of free-living amebas




 Ulceration of the skin containing both amebic trophozoites and cysts suggests also the portal of




 entry into the bloodstream. In experimental animals, the olfactory neuroepithelium has also been




 found to be a possible portal of entry (Janitschke et al, 1996). The incubation period of GAE is




 unknown but is probably longer than 10 days. The ability of the Acanthamoeba to produce




 necrosis of the brain tissue is probably due to an enzymatic action induced by lysosomal




 hydrolases and phospholipase that can degrade phopholipids of the myelin sheaths (Martinez and



 Visvesvara, 1997).









 Studies in mice have demonstrated that it is possible to immunize animals against Acanthamoeba




 meningoencephalitis (Culberton, 1971; Rowan-Kelly and Ferrante, 1984). Animals immunized




 intraperitoneally with sonicated trophozoites of A. culbertsoni were highly resistant to intranasal




 infection with the organism. Those immunized with a non-pathogenic A. culbertsoni or A.




polyphaga were not protected against infection with A. culbertsoni.
5.3 GAE in Domestic Animals and Wildlife




Several reports of arnphizoic amoebae in animals appeared in the literature at about the,same



tune as they were found in fatal infections in humans. The principal difference between human
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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 niiost extensive surveys of infection in domestic




animals by amphizoic amoeba. He identified Acanthamoeba spp. from bulls, cows, a rabbit,




pigeons and turkeys. Infections in animals probably occur by the same routes as reported for




humans. It has also been described in dogs by several investigators (Ayers et al., 1972, Bauer et




al, 1993). Infections in the lung of water buffalo and bulls could have been nasopharyngeal




from drinking unclean water (Dwivedi and Singh, 1965, McConnell et al, 1968).








Evidence for water as a source of infection in animals by Acanthamoeba is found in reports of




the amoebae in the gills, spleen, urinary bladder or blood of wild caught and ornamental fish




(Taylor, 1977, Dykova et al., 1996, Booton et al., 1999).








5.4 Other Infections caused by Acanthamoeba




Occasional infections by Acanthamoeba spp. have included a purulent discharge from an ear




(Lengy et al., 1971), a granulomatous skin lesion (Gullet et al., 1979), rhinosinusitis hi an AIDS




patient (Teknos et al., 2000) and possible association with intestinal disorders (Hoffler and




Rubel, 1974; Mehta and Guirges, 1979; Thamprasert et al, 1993).
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 5.5 Immunocompromised Individuals



 Several reports of Acanthamoeba infection in ADDS patients involved the skin, as well as other

                  t

 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 a/.(1987) examined a 34 year-old patient



 with a history of nasopharyngeal allergies and infections with Giardia lamblia and
                  r  '                                        •


 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 a/.(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).
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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
                                          I
received bone marrow transplants.            .

                                          i

                                          i
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).



5.7 Effect of Endosvmbiosis on Virulence

Acanthamoeba spp.has been demonstrated to develop endosymbiotic relationships with a number

 of waterborne bacteria, including Legionella pneumophila and Mycobacterium avium ( Table

 3.3). This relationship may be important both in the growth and survival of these opportunistic

 pathogens in drinking water systems, and in their ability to cause disease in humans.
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 Cirillo et al. (1997) found that Mycobacterium avium replicates v/itiwiAcanthamoeba castellanii
           ''                                                       «•
 and that this association enhanced both the entry and intracellular replication compared to the

 growth of the bacteria in broth culture. Furthermore, amoeba-grown M avium was also more

 virulent in a mouse model. They also found that the highest growth rate of the M. avium in the

 amoebae was  near 37°C. From this observation, they suggested that if growth of M avium in

 water environments occurs primarily within protozoa, the fact that M.  avium has 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
                  I
 Mycobacterium were readily killed within the amoeba.
 Cirillo et al, 1999 found Legionellapneumophila grown hi 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
                  i                                            -     '              -
 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
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Acanthamoeba infections is unknown.
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                              6.0 RISK ASSESSMENT

6.1 The Organisrii and its Occurrence fExposurel
                 i                   '                 '   .
Certain species of the genus Acanthamoeba have been associated with eye disease hi 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.
                 I
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 bv
Tapwater

Molecular based investigations have established domestic tapwater in the United Kingdom as a
                 i
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 hi the midwest United States suggested that an

epidemic of presumed Acanthamoeba infections was associated with municipal water supplies
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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

                                          i
from a private well.  In both of these studies th|e 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.
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           Table 6.1 Human Infection Caused by Species of Acanthamoeba
  Species of
  Acanthamoeba
CNS       Eye       Other tissues
infection   infection
                              Reference
 A. astronyxis
 A. castellanii
 •A. culbersoni
X
X
          X
X
           Adrenal, lymph
           node, sinus, skin,
           thyroid

           Lung, prostate,
           bone, muscle,
           sinus, skin
Liver, spleen,
uterus, skin
                   Gullett et al. (1979)
Martinez (1982)
Martinez et al. (1977)
Moore et al (1985)
Borochovitz et al. (1981)
Gonzalez et al. (1986)

Martinez et al (1977)
Wiley et al (1987)
Mannisefa/. (1986)
May et al (1992)
A. divionensis
A.grifflni
A. hatchetti
A. healyi
A. palestinensis
A. polyphaga
A. rhysodes
X


X
;X
'••••-.
X

X
X


X
X
DiGregorio (1992)
Ledeee/a/. (1996)
Cohen et al. (1985)
Kim et al. (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 fj,m) and
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cysts (15 to 28 //m) they would be easily removed by filtration in a conventional water treatment

                                           i
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


iodine. The chlorine resistance of two different strains varied considerably. A 99.99% (4 Iog10)


inactivation of a more sensitive strain was achieved with 16 mg/liter within one hour. A 4-log,0


decrease was not achieved after 24 hours with 6 nig/liter.





The cysts have also been found to be very resistant to ultraviolet light.  Chang 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 log,0) inactivation of the cysts. The


viability of the cysts was detected with a plaque assay on a lawn of Escherichia coli bacteria,
                                           i

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
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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
                 r
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 Legionetta pneumophila

and Mycobacterium avium.



6.4 Dose Response

Badenoch et al. (1990) demonstrated Acanthamoeba infections could be induced in the rat

cornea by co-inoculation with the bacterium Corynebacterium xerosis. The co-inoculation with

C. xerosis was necessary to induce the Acanthamoeba infection.  Infection resulted in 7 of 24 rats

that were exposed to 103 trophozoites and 1 in 10 animals when exposed to 104 trophozoites. At

least 104 C. xerosis had to be co-inoculated to achieve these infection rates. The results suggest
                 i
that at least 103 trophozoites are necessary to cause Acanthamoeba eye infection.
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  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
Useoftapwater
 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.
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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 hi




contact lens wearers: can be eliminated by proper care of the lens to avoid exposure 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 lenses 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 Mycobacteriumavium 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 Acanthamoeba 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. The 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
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may have become contaminated enhanced the risk of presumed Acanthamoeba keratitis (Meier et
al, 1998). Seasonal distribution of keratitis and abundance of Acanthamoeba in surface waters
also suggests a relationship. Additional information on dose needed for infection and
quantitative data on occurrence in drinking water supplies would help to better understand the
potential risks to contact lens wearers and the general public.  The incidence of recognized
Acanthamoeba keratitis is around 1-2/106 (Table 5.3). The highest incidence in the U.S., which
may have been likened to flooding and the use of municipal water supplies, was 14/106 (Meier et
al, 1998). Even if all the cases of Acanthamoeba were associated with tapwater this would be
                                          i
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).
             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)	
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 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 late 1970's gas permeable hard lenses (which allow

more oxygen to reach the eye) were developed.



                  Table 7.1 History of Contact Lens Development1
 Year
Event
 1887
 1939
 1948
 1971
 1978
 1981

 1986
 1987
First contact lens made from glass; covers the entire eye
Contact lenses first made from plastic
Plastic contact lenses designed to cover the cornea only
Introduction of soft contact lenses
Introduction of oxygen permeable lenses
Food and Drug Administration approves soft contact lenses for
extended (overnight) wear
Overnight wear oxygen permeable lenses become available
Introduction of disposable soft contact lenses	
 1 Source: Contact Lens Council, 2000-
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The Food and Drug Administration must approve all contact lenses before they are available to
                                          i
the public. The types of contact lenses currently in use are listed in Table 7.2.



                          Table 7.2 Types of Contact Lenses
 Type
   Comments
 Daily wear soft lenses



 Daily wear disposable soft lenses

 Extended wear soft lenses



 Extended wear disposable soft lenses



 Rigid gas permeable lenses
   Made of soft, flexible plastics that allow
   oxygen to pass through to the eye
   Cleaning is required

   Typically no lens care is required

   Available for overnight wear
   Can usually be prescribed for up to seven days
   of wear without removal

   Worn from one to six nights and then
   discarded
   Require little or no cleaning

   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
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permeable. Most wearers use daily wear lenses which are removed at bedtime, while 25% use

extended wear lenses (Table 7.3).

                  Table 7.3 Wearers and Types of Contact Lenses1
                       Type of lens
               Percent of wearers
                       Soft lenses           85
                       Rigid gas permeable   15
                       Daily wear           75
                       Extended wear        25
                            1 Source: Contact Lens Council
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.
      Table 7.4 Age Distribution of Contact Lens Wearers in the United States1
 Age (years)
% of soft contact lens wearers   % of rigid gas permable
                            contact lens wearers
 18 to 25
 26 to 39
 >40
10
23
45
22
3
10
26
61
1 Source: Contact Lens Council, 2000
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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.

          Table 7.5 Risk Factors Associated with Acanthamoebic keratitis
     Risk Factor
          % of Acanthamoebic
          keratitis cases
    Wore contact lenses
    Wore daily wear lenses                j
    Wore extended wear lenses
    History of corneal trauma              |
    History of exposure to contaminated tapwater
          85
          56
          19
          26
          25
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 hi storage cases. Any contact lens is a

potential carrier ofAcanthamoeba to the eye surface after being exposed to a contaminated fluid.
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        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 perrneable
Illingworth et
al.,1995
21
67
8
4
Percentage of cases
Stehr-Green et aL,
1987
56
19
2
7
Moore et aL,
1985
75
14
6
4
The use of non-sterile solutions such as tap water, bottled water and non-sterile distilled water

have been associated \viihAcanthamoeba infections among contact lens wearers (Moore et aL,

1985; Stehr-Green et aL, 1987).



    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
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-

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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

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 hi a greater risk of keratitis because of Acanthamoeba

resistance to this disinfectant (Illingworth et al., 1995).



7.4 Contact Lens Disinfection
                                          i
                                          i
7.4.1 Studies of Lens Disinfection

Procedures for disinfecting different types of contact lenses and lens equipment have been

investigated (Knoll, 1971). Newer and safer methods for lens care were proposed by the U.S.

Food and Drug Administration (1973) even before contact lens-associated amoebic keratitis was

discovered. Busschaert et al. (1978) had found that moist heat sterilization, 80°C for 10 minutes,

provided an adequate margin of safety for disinfecting hydrophilic contact lenses. Acanthamoeba

readily adheres to contact lenses. The degree of adherence depends on water content, surface

tension and surface charge (Gorlin et al, 1996). Kilvington (1989) investigated the killing

 capacity of moist heat against cysts of A. polyphaga, which survived a contact time of 60

 minutes at 50°C to 60°C, but were inactivated when temperature was increased to 65°C to 70°C.
                                          i
 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
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 cleaned periodically, they probably should be replaced at some frequency to avoid a build up of




 debris and contaminating microorganisms.  ,
 Brandt et al. (1989) tested saline solutions, cleaning solutions, and disinfection solutions against




 three species of Acqnthamoeba recovered from contact lens cases, i.e., A. castellanii, A.




 culbertsoni, and A. polyphaga.  Although solutions containing hydrogen peroxide were the most




 effective, cysts were detected in all solutions for at least 6 hours after treatment.  The authors




 concluded that, at the time of their study, none of the solutions available on the market were




 effective for eliminating cysts of Acanthamoeba within a short period of disinfection. Silvany et




 al (1990) tested A. castellanii ATCC 30868 and A. polyphaga ATCC 30873 against 13




 commercially available solutions.  Growth occurred within as few as 30 minutes after exposure




 to one solution, with growth inhibited for up to 24 hours with five others. Two solutions




 containing hydrogen peroxide and three containing chlorohexidine inhibited growth within 30




 minutes; one solution containing benzalkonium chloride inhibited growth within 1 hour. In this




 study and others (Brandt et al, 1989), it was concluded that, at that time, there was neither one




 solution nor one treatment protocol that was effective against all species of Acanthamoeba.




Rutherford et al (1991) tested chlorhexidine in tablet form to find a procedure that would require




 less time for cleaning and disinfection. They tested a tablet dissolved hi potable water for




 amoebicidal activity against trophozoites and cysts of A. castellanii and A. polyphaga isolated




from human corneas, and against A castellanii ATCC 30010. None of the amoebae excysted




and grew after exposure times of 4,6, 8,  and 24 hours. Results showed that soft contact lenses
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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. potyphaga: hydrogen
                                            i       .  '                       •     '

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 used did not. Although chlorheximide inactivated 1x10 cysts down to


approximately 1x10* 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 comeal


 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 hi 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
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 morning, a one step product has been produced, based on adding a neutralizing tablet to the

 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).
                  h
 This is insufficient time to kill microbes on the lens.
 7.4.3 Multi-Purpose Solutions


 Due to problems with hydrogen peroxide, multi-purpose solutions have been produced to clean

 and store lenses with a single solution without the need for neutralization. This is achieved by

 combining a poloxomer (detergent) with a chemical disinfectant (PHMB) or polyquaternium

 with appropriate buffers and EDTA. It is provided as a sterile solution in sufficient quantity for

 rub and rinse cleaning and storing of the lenses and washing of the storage case. Products may

 contain from 0.5 to 5ppm of PHMB. The lower concentration is less effective against bacteria

 and has no activity against Acanthamoeba. At this low concentration, eradicating Acanthamoeba

 depends on cleaning by the rinse and rub technique. The higher concentration is most effective

 against bacteria and fungi and is also acanthamoebicidal for 102 cysts (Seal et al, 1992).

 Similarly, polyquaternium is used at low concentrations that have poor bactericidal activity and

 no acanthamoebicidal activity. Multipurpose solutions provide the easiest technique for the lens

 wearer to clean and disinfect the lens, and give better compliance results. The main advantage of

 these solutions is that the product is sterile, and there is no need to wash the storage case with tap

 water. The poloxomers used have a good surfactant action for removal of microbes adhering to

the lens. Provided the storage case is changed monthly and tap water contamination is avoided,
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these solutions represent the most user friendly method, Bactericidal activity is reasonable, but
                                          I
not the best. Use of solutions with PHMB as the disinfectant at a minimum concentration of 5

ppm gives an enhanced microbiocidal effect, including activity against Acanthamoeba.



Hiti et al, 2001 recently reported the use of microwaves to inactivate contact lenses

contaminated with acanthamoeba. Different types of contact lens cases were contaminated with

trophozoites and cysts of three different Acanthamoeba species (A. comandoni, A, castellanii,

and A. hatchetti) and were exposed to microwave irradiation for various periods of time.

Trophozoites, as well as cysts of the different Acanthamoeba strains, were effectively killed,

even by only 3 minutes of microwave irradiation, and there were no negative effects of

irradiation on the contact lens cases themselves.
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                                   8.0 DATA GAPS

Risk fromAcanthamoeba keratitis is complex depending upon the virulence of the particular
                  i
                  !
strain, exposure, trauma or other stress to the eye and host immune response. Bacterial
                  i
endosymbionts may also play a factor in pathogenicity of Acanthamoeba. Which factors) may

be the most important is not clear. The recent work of Alizadeh et al., (2001) suggests that the

ability of the host to produce IgA antibodies may be a significant factor. Thus, immune response
                  »
could be a deciding factor as it appears in GAE infection and AIDS patients. If so then a certain

sub-population with an inability to produce IgA in the tears may be at greatest risk.




No data could be found on the occurrence or types of Acanthamoeba in tapwater in the United

States. Published work on presence in tapwater does not provide information on the type of

treatment the water received or the level of residual chlorine. Assessment of the pathogenicity by

cell culture and molecular methods of Acanthamoeba in tapwater would also be useful in the risk
                  i                   ' '
assessment process for drinking water.



The possibility that Acanthamoeba spp. might serve as vectors for bacterial infections from water
                  i                        _                         *
sources also needs to be explored. The bacterial endosymbionts include an interesting array of

pathogens including Vibrio cholerae andLegionellapneumophila, both of which are well

recognized water-borne/water-based pathogens. Work is needed to  determine if control of

Acanthamoeba spp; is needed to control water-based pathogens in water supplies.
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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
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                                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, RJ. and Banta, 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.
                  i
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. OphthainoL 19:9f20.

Badenoch, P.R., Johnson, AM., Christy, P.E., and Coster, DJ.  1990. Pathogenicity of
Acanthamoeba and a Corynebacterium in the . Arch. Ophthalmol. 108:107-112.

Bauer, R.W., Harrison, L.R., Watson, C.W., Styer, EX., 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, TJ. 1999.
Morphological and fDNA similarities of Acanthamoeba strains parasitic in fish and those
 causing human disease. J. Eukaryotic Microbiol. 46:6A.

 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.
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