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

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

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

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

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

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

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

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

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

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

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

Management
support
was
provided
by
Geoffrey
Grubbs,
Director,
Office
of
Science
and
Technology,
Office
of
Water,
U.
S.
EPA.
Health
Effects
Support
Document
for
Acanthamoeba
iii
TABLE
OF
CONTENTS
LIST
OF
TABLES
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LIST
OF
FIGURES
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GLOSSARY
OF
TERMS
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vii
1.0
EXECUTIVE
SUMMARY
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1­
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2.0
INTRODUCTION
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2­
1
3.0
GENERAL
INFORMATION
AND
PROPERTIES
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3­
1
3.1
History
and
Taxonomy
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3­
1
3.2
General
Characteristics
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3­
2
3.3
Methods
of
Identification
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3­
5
3.4
Cultivation
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3­
5
3.5
Significance
of
Endosymbiosis
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3­
5
4.0
OCCURRENCE
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4­
1
4.1
Water
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4­
2
4.1.1
Surface
Waters
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4­
2
4.1.1.1
Freshwaters
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4­
2
4.1.1.2
Seawater
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4­
2
4.1.2
Tapwater
and
Bottled
Water
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4­
3
4.1.3
Swimming
Pools
and
Spas
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4­
4
4.1.4
Sewage
and
Biosolids
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4­
4
4.2
Animal
Wastes
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4­
5
4.3
Air,
Dust
and
Soil
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4­
5
4.4
Summary
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4­
5
5.0
HEALTH
EFFECTS
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5­
1
5.1
Eye
Infections
(
Acanthamoebic
Keratitis)
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5­
3
5.1.1
Symptoms
of
Acanthamoeba
Keratitis
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5­
5
5.1.2
Diagnosis
of
Acanthamoeba
Keratitis
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5­
6
5.1.3
Identification
Procedures
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5­
6
5.1.4
Treatment
of
Acanthamoebic
Keratitis
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5­
6
5.1.5
Incidence
of
Acanthamoeba
Keratitis
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5­
7
5.1.6
Pathogenicity
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5­
8
5.1.7
Immunity
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5­
9
Health
Effects
Support
Document
for
Acanthamoeba
iv
5.2
Granulomatous
Amoebic
Encephalitis
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5­
10
5.2.1
Diagnosis
and
Treatment
of
GAE
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5­
12
5.2.2
Incidence
of
GAE
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5­
12
5.2.3
Pathogenesis
and
Immunity
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5­
13
5.3
GAE
in
Domestic
Animals
and
Wildlife
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5­
13
5.4
Other
Infections
caused
by
Acanthamoeba
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5­
13
5.5
Immunocompromised
Individuals
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5­
14
5.6
Incidence
to
Children
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5­
14
5.7
Effect
of
Endosymbiosis
on
Virulence
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5­
15
6.0
HEALTH
EFFECTS
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6­
1
6.1
The
Organism
and
its
Occurrence
(
Exposure)
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6­
1
6.2
Epidemiological
Evidence
for
Acanthamoeba
Keratitis
Transmission
by
Tapwater
6­
1
6.3
Resistance
to
Drinking
Water
Treatment
and
Disinfection
.
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6­
2
6.4
Dose
Response
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6­
3
6.5
Risk
Characterization
.
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.
6­
3
7.0
ASSOCIATION
OF
CONTACT
LENSES
WITH
ACANTHAMOEBIC
KERATITIS
.
.
.
7­
1
7.1
Types
of
Contact
Lenses
.
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.
7­
1
7.2
Demographics
of
Contact
Lens
Use
.
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7­
2
7.3
Risk
Factors
.
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7­
3
7.4
Contact
Lens
Disinfection
.
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7­
5
7.4.1
Studies
of
Lens
Disinfection
.
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7­
5
7.4.2
Hydrogen
Peroxide
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7­
6
7.4.3
Multi­
Purpose
Solutions
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7­
7
8.0
DATA
GAPS
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8­
1
9.0
REFERENCES
.
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.
9­
1
Health
Effects
Support
Document
for
Acanthamoeba
v
LIST
OF
TABLES
Table
2.1
Major
Waterborne/
Water­
based
Pathogenic
Protozoa
.
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2­
1
Table
3.1
Currently
Identified
Species
of
Acanthamoeba
.
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3­
1
Table
3.2
Acanthamoeba
Species
Classification
.
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.
3­
2
Table
3.3
Bacterial
Endosymbionts
of
Acanthamoeba
.
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3­
6
Table
4.1
Occurrence
of
Acanthamoeba
.
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4­
1
Table
5.1
Comparison
of
Clinical
and
Pathological
Features
of
Granulomatous
Amoebic
Encephalitis
(
GAE)
and
Acanthamoeba
Keratitis
(
AK)
.
.
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.
5­
1
Table
5.2
Characteristics
and
Symptoms
of
Patients
with
Acanthamoeba
Keratitis
.
.
.
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.
5­
3
Table
5.3
Worldwide
Incidence
of
Acanthamoeba
Keratitis
.
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5­
8
Table
6.1
Human
Infection
Caused
by
Species
of
Acanthamoeba
.
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6­
2
Table
6.2
Mechanisms
involved
in
Acanthamoeba
Keratitis
.
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.
6­
4
Table
7.1
History
of
Contact
Lens
Development
.
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7­
1
Table
7.2
Types
of
Contact
Lenses
.
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.
7­
2
Table
7.3
Wearers
and
Types
of
Contact
Lenses
.
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.
7­
3
Table
7.4
Age
Distribution
of
Contact
Lens
Wearers
in
the
United
States
.
.
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.
.
7­
3
Table
7.5
Risk
Factors
Associated
with
Acanthamoebic
Keratitis
.
.
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.
7­
4
Table
7.6
Types
of
Contact
Lenses
Associated
with
Acanthamoebic
Keratitis
.
.
.
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.
.
7­
4
Table
7.7
Risk
Factors
for
Acanthamoebic
Keratitis
in
Contact
Lens
Wearers
.
.
.
.
.
.
.
.
.
7­
5
Health
Effects
Support
Document
for
Acanthamoeba
vi
LIST
OF
FIGURES
Figure
3.1
Life
Cycle
of
Acanthamoeba
Species
.
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.
3­
3
Figure
3.2
Acanthamoeba
trophozoite.
.
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3­
4
Figure
3.3
Cysts
of
Acanthamoeba.
.
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.
3­
4
Figure
3.4
Significance
of
Endosymbiosis
to
Waterborne
Disease
Transmission
.
.
.
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.
.
.
.
3­
7
Figure
5.1
Life
Cycle
of
Acanthamoeba
spp.
and
Human
Infection
.
.
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.
5­
2
Figure
5.2
Slit
lamp
view
showing
a
paracentral
complete
ring
infiltrate
of
the
cornea
.
.
.
.
5­
5
Figure
5.3
Normal
Eye
.
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.
5­
5
Figure
5.4
Granulomatous
Amoebic
Encephalitis
(
GAE)
.
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.
.
5­
11
Figure
6.1
Eye
Trauma
and
Contact
Lenses
as
Determinants
of
Susceptibility
to
Acanthamoeba
Keratitis
.
.
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.
6­
5
Health
Effects
Support
Document
for
Acanthamoeba
vii
GLOSSARY
OF
TERMS
Amphizoic
amoeba
Amoeba
able
to
live
both
free
in
nature
and
as
pathogens
in
a
host
Anterior
uveitis
Inflammation
of
the
iris
and
ciliary
body
Axenic
Grown
in
the
absence
of
other
microorganisms
Cytopathogenic
effects
Alteration
of
the
appearanc
of
animal
cells
in
culture
due
to
the
growth
of
pathogenic
microorganisms
Confocal
microscopy
Microscopy
using
a
laser­
scanning
fluorescent
microscope
which
gives
a
digital
two­
dimensional
signal
that
is
reconstructed
into
a
three
dimensional
image
Cornea
The
clear,
transparent
anterior
portion
of
the
fibrous
coat
of
the
eye
Endocyst
The
innermost
cellulose­
containing
layer
of
the
Acanthamoeba
cyst.
It
may
be
stellate,
polygonal,
oval,
triangular,
or
round.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Table
3.3
Bacterial
Endosy
mbionts*
of
Acanthamoeba
Legionella
pneumophila
Mycobacterium
avium
Burkholderia
picketti
Vibrio
cholerae
Francisella
tularensis
Chlamydia
pneumoniae
Rickettsiales
Listeria
monocytogenes
Fritsche
et
al.,
1999;
Ly
and
Miller,
1990
*
live
within
the
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Figure
3.4
Significance
of
Endosymbiosis
to
Waterborne
Disease
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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
Crespo
et
al.,
1990
Tap
water
(
Mexico)
Rivera
et
al.,
1979
Bottled
water
(
Mexico)
Rivera
et
al.,
1981
Hospital
tap
water
Rohr
et
al.,
1998
Eyewash
stations
Tyndall
et
al.
,
1987
Freshwater
ponds
John
and
Howard,
1995
Thermal
water
DeJonckheere,
1979,
Dive
et
al.,
1982
Well
water
Jones
et
al.,
1975
Physiotherapy
tubs
Penas­
Ares
et
al.,
1994
Aquaria
DeJonckheere,
1979
Municipal
sewage
Singh
and
Das,
1972
Ocean
sewage
dump
site
Sawyer
et
al.,
1982
House
dust
Yamaura
et
al.,
1993
Garden
soil
Singh,
1952
Sand
box
Yamaura
et
al.,
1993
Garden
vegetables
Rude
et
al.,
1984
Fish
Taylor,
1977
Air
conditioner
Walker
et
al.,
1986
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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
in
this
study,
but
was
recovered
only
on
three
occasions
and
did
not
exceed
a
population
of
200/
g.
Detterline
&
Wilhelm
(
1991)
collected
water
samples
from
59
sites
in
federally
managed
recreational
waters
of
the
U.
S.
and
recovered
temperature­
tolerant
strains
of
Acanthamoeba
from
16
of
31
sites
that
grew
at
37
º
C.
Kyle
and
Noblet
(
1987)
published
a
detailed
account
of
amoebae
present
in
a
spillway
reservoir
in
South
Carolina.
The
authors
studied
the
lake
throughout
the
course
of
a
year
to
record
seasonal
influences
on
amoeba
populations,
such
as
dissolved
oxygen,
attenuation,
and
water
temperature.
Information
on
amphizoic
amoebae
from
this
study
showed
that
in
the
surface
water
they
ranged
from
5
to
10
amoebae
/
50
milliliters
(
ml)
water
in
May,
and
peaked
at
98/
50
ml
in
July.

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

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
sewagecontaminated
inshore
New
York
and
New
Jersey
shellfish
beds
that
periodically
were
closed
to
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shellfish
harvesting.
Munson
(
1993)
recovered
several
species
of
Acanthamoeba
from
coastal
waters
of
Bermuda,
and
noted
a
high
frequency
of
recovery
of
Acanthamoeba
spp.
near
sewage
outfalls.

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

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

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

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

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

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

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

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

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

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

5.1
Eye
Infections
(
Acanthamoeb
ic
Keratitis)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

A.
divionensis
X
DiGregorio
(
1992)

A.
griffini
X
Ledee
et
al.
(
1996)

A.
hatchetti
X
Cohen
et
al.
(
1985)

A.
healyi
X
Kim
et
al.
(
2000)

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

A.
polyphaga
X
Singh
and
Petri
(
2000)

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

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

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

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

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

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

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

While
it
is
clear
that
a
relationship
exists
between
Acanthamoeba
in
water
and
keratitis,
the
role
of
tapwater
is
not
clearly
understood.
Data
on
the
occurrence
and
concentration
of
Acanthamoeba
in
the
United
States
is
lacking.
One
study
suggests
that
municipal
studies
which
may
have
become
contaminated
enhanced
the
risk
of
presumed
Acanthamoeba
keratitis
(
Meier
et
al.,
1998).
Seasonal
distribution
of
keratitis
and
abundance
of
Acanthamoeba
in
surface
waters
also
suggests
a
relationship.
Additional
information
on
dose
needed
for
infection
and
quantitative
data
on
occurrence
in
drinking
water
supplies
would
help
to
better
understand
the
potential
risks
to
contact
lens
wearers
and
the
general
public.
The
incidence
of
recognized
Acanthamoeba
keratitis
is
around
1­
2/
106
(
Table
5.3).
The
highest
incidence
in
the
U.
S.,
which
may
have
been
likened
to
flooding
and
the
use
of
municipal
water
supplies,
was
14/
106
(
Meier
et
al.,
1998).
Even
if
all
the
cases
of
Acanthamoeba
were
associated
with
tapwater
this
would
be
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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).
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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
Fick.
The
modern
contact
lens
was
developed
in
1948,
and
is
made
of
plastic
and
rests
on
a
cushion
of
tears
(
Table
7.1).
It
covers
the
cornea
approximately
over
the
iris
and
pupil.
The
hard
plastic
contact
lenses
had
a
limited
wearing
time
because
of
potential
irritation
of
the
cornea.
In
the
1970'
s,
soft
lenses,
made
from
water
absorbing
plastic
gel
for
greater
flexibility,
were
introduced.
In
the
1980'
s
extended
wear
soft
lenses,
which
can
be
worn
without
removal
for
several
weeks
at
a
time,
were
introduced.
Soft
contact
lenses
are
usually
more
comfortable
because
they
allow
oxygen
to
penetrate
to
the
surface
of
the
eye.
In
the
1970'
s
gas
permeable
hard
lenses
(
which
allow
more
oxygen
to
reach
the
eye)
were
developed.

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

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

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

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

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

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

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

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

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

Hiti
et
al.,
2001
recently
reported
the
use
of
microwaves
to
inactivate
contact
lenses
contaminated
with
acanthamoeba.
Different
types
of
contact
lens
cases
were
contaminated
with
trophozoites
and
cysts
of
three
different
Acanthamoeba
species
(
A.
comandoni,
A,
castellanii,
and
A.
hatchetti)
and
were
exposed
to
microwave
irradiation
for
various
periods
of
time.
Trophozoites,
as
well
as
cysts
of
the
different
Acanthamoeba
strains,
were
effectively
killed,
even
by
only
3
minutes
of
microwave
irradiation,
and
there
were
no
negative
effects
of
irradiation
on
the
contact
lens
cases
themselves.
Health
Effects
Support
Document
for
Acanthamoeba
8­
1
8.0
DATA
GAPS
Risk
from
Acanthamoeba
keratitis
is
complex
depending
upon
the
virulence
of
the
particular
strain,
exposure,
trauma
or
other
stress
to
the
eye
and
host
immune
response.
Bacterial
endosymbionts
may
also
play
a
factor
in
pathogenicity
of
Acanthamoeba.
Which
factor(
s)
may
be
the
most
important
is
not
clear.
The
recent
work
of
Alizadeh
et
al.,
(
2001)
suggests
that
the
ability
of
the
host
to
produce
IgA
antibodies
may
be
a
significant
factor.
Thus,
immune
response
could
be
a
deciding
factor
as
it
appears
in
GAE
infection
and
AIDS
patients.
If
so
then
a
certain
sub­
population
with
an
inability
to
produce
IgA
in
the
tears
may
be
at
greatest
risk.

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

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

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.
Health
Effects
Support
Document
for
Acanthamoeba
9­
1
9.0
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