1.

03
Free Living Pathogenic Amebae

June 19, 2015

Dr. Nacpil

OUTLINE
I.

II.

Naegleria sp.
a. Biology
b. Species
c. Multiplication and Life Cycle, Epidemiology
d. Pathogenesis
e. Clinical Manifestations
f. Diagnosis
g. Treatment
h. Prevention and Control
Acanthamoeba
a. Biology
b. Multiplication and Life Cycle, Epidemiology
c. Pathogenesis
d. Clinical Manifestations
e. Diagnosis
f. Treatment
g. Prevention and Control

Naegleria sp.

Distinguished from N. fowleri through cell morphology,

culture medium preferences, temperature tolerance,
lectin sensitivity, isozyme pattern, DNA restriction
patterns, mouse pathogenicity and serology.

Naegleria philppinenesis

Locally occurring species

Recorded to be pathogenic in mice

Naegleria fowleri

Pathogenic species

Agent of Primary Amebic Meningoencephalitis (PAM)

Name from Malcolm Fowler, who first isolated the

organism from a patient
Other notable species: (from Belizario)

N. australiensis (isolated from Australia)

N. australiensis subsp. Italica (same species as above

but isolated from Europe)
Multiplication and Life Cycle, Epidemiology
Life Cycle

Naegleria spp. In cyst, trophozoite and flagellate form ( left to right)

Is free living amebo-flagellate, because the organism is

capable of existing as an ameba (Trophozoite form) and
as a flagellate (Swimming form)
Can also transform to a resistant cyst stage
Is the primary causative agent of the Primary Amebic
Meningoencephalitis (PAM)
Biology

Trophozoite

With a characteristic lobular monopseudopodium

Prominent nucleus with dense central nucleolus

Cytoplasm is finely granular with numerous food

vacuoles

Infective and Pathogenic Stage of the parasite

Reproductive stage of the parasite (via Promitosis)

o Promitosis form of binary division in which
nuclear membrane remains intact

In nature, trophozoite will transform into biflagellated form

May encyst if conditions are not favorable for growth

Cyst

8-12 um in diameter
Smooth single-layered wall
Single nucleus with dense central nucleolus
Presence of 1-2 cyst pores
o Cyst pores mucus plugged pores in which
trophozoite will emerge

Flagellated Form

Pear-shaped body with a pair of flagella in one of the tips

Can transform from trophozoite stage within 2-3 hours or

up to 3-4 days.

Not found in human tissue, occasionally found in CSF

Species
Naegleria gruberi

Non-pathogenic, most commonly studied

Utilized in morphogenetic studies on differentiation and

induction of flagellation, along with the origin of flagellar
apparatus

Is a free living amebae found in fresh water and soil, but

may tolerate hyperchlorinated water up to 0.5 ug/mL
o
Thermophilic organism up to 46 C
Reproduces via binary division (written here as
promitosis)
Trophozoite is the reproductive stage. Cyst and
Flagellate form are non-reproductive
May encyst if conditions are unfavorable

Epidemiology

Soil is the preferred habitat

Isolated samples from:

o chlorinated swimming pools
o freshwater lakes
o thermal springs
o domestic water supplies
o sewage
o soil
o air humidifier system
o human throat and nasal cavities
o air conditioning systems

In the Philippines, samples were isolated from:

o Moist soil
o Fresh water habitat

Free Living Pathogenic Amebae

o Industrial and Natural thermally polluted rivers

Other samples isolated are from:
o Australia ( N. australiensis pathogenic only to
mice)
o Europe ( N. australiensis subsp. Italica)
Earliest Reports of Diseases:
o (1951 1952) PAM epidemic in Richmond,
Virginia noted by Dos Santos
o (1965) PAM case in South Australia noted by
Fowler and Carter
o (1966) PAM in Texas by Patras and Andujar
o (1968) PAM in Florida by Butt et. al
o (No Date) One case of PAM in the Philippines
Pathogenesis

Infection parasite may enter the body either by being

inhaled or through contact with the olfactory epithelium.
Sustentacular cells of the olfactory epithelium are
capable of active phagocytosis (Mechanism in which
amoeba invades the body.
o Note: The organism may also go down to other
organs such as the GIT or the lungs, although
this rarely causes diseases.
The parasite makes it way to the olfactory bulb in the
arachnoid space through the unmyelinated olfactory
nerves.
The arachnoid space become the route of dissemination
into the CNS

Has both in vitro and in vivo activity against N.

fowleri

Non-Pharmacologic

Free Chlorine
o Cysts are killed at 5ug/mL
o Trophozoites killed at lower doses

Elemental Iodine
o At greater than 3.4ug/mL, more cysticidal than
free chlorine

Chlorine, Chlorine dioxide, Deciquam 22, Baquacil

o Amebicidal properties based on physical and
chemical properties of water treated

Cyanuric Acid
o N. gruberi cysts are very susceptible to this

0.2% NaCl and KCl

o Inhibits growth

CaCl2
o Stimulate encystment
Other Treatment Forms

Temperature
o N. fowleri does not tolerate temps higher than
100oC and lower than 65oC

Drying
o Lethal to trophozoites
o Cysts remain viable within 23 months

Lyophilisation
o N. fowleri cysts are rendered non-viable
o N. gruberi cysts remain viable after 6 months

Clinical Manifestations
Prevention and Control
Gastritis and Diarrhea (rare, only when parasite goes to GIT)
Pneumonitis (rare, only when parasite goes into lungs)
Primary Amoebic Meningoencephalitis (PAM)

Affects children and young adults

Incubation period 2-3 days or 7-15 days

S/Sx include:
o Sudden onset of bitemporal or bifrontal
headache
o Fever, vomiting, stiff neck
o Rapid progression to lethargy or coma
o Death within 48-72 hours

No means of prevention and control

Avoid swimming in warm and stagnant freshwater pools,
water discharge pools, unchlorinated poorly maintained
swimming pools or mud-lined lakes and ponds
Acanthamoeba sp.

Diagnosis
Actual presence of organism and CSF

Was done previously

Aspirate from suspected infection may be placed in

bacteria seeded agar and exhibit trophozoites in 24 hours

Naegleria usually identified through there blunt lobose

pseudopodia and directional motility.
PCR and ELISA

Currently used for specific N. fowleri tests

Can be used for amebae with negative flagellation

(Benets, 2003)
Treatment
Pharmacologic (DOC is Ampotericin B)

Amphotericin B
o Produce changes in nucleus and mitochondria
of amoeba
o Increase proliferation of RER and SER
o Decrease food vacuoles
o Increase formation of autophagic vacuoles
o Inhibit pseudopod formation
o Induce blebbing of amoeba plasma membrane
o Associated with adverse effects on kidney and
organs

Clotrimoxazole
o Amoebicidal activity in vitro but effect in in vivo

Tremethoprim
o Prevent folic acid metabolism
o Effective in preventing growth of non-virulent
strains but not in virulent strains

Actinomycin
D,
Daunomycin,
Mithramycin,
Sulfamethoxazole, Tyrocidine
o N. fowleriis is less resistant to these when
grown in Balamuth medium than when grown in
Nelson medium

Azithromycin
TRANSCRIBERS: PARAng Pag-ibig

Acanthamoeba sp. cyst (left), trophozoite (right)

Small, free-living amoeba characterized by an active

trophozoite stage and a dormant cyst stage
Sluggishly motile trophozoites feed on gram-negative
bacteria, blue-green algae or yeasts
Reproduce by binary fission and encyst if environment is
unfavourable
Has small locomotory filaments known as acanthopodia
Ubiquitous organism
Biology

Cyst

Double-walled outer wrinkled wall; inner polygonally

shaped wall
Pores or ostioles are seen at the point of contact
between the two walls
Nucleus with a large dense central nucleolus
surrounded by a clear halo

Trophozoite

Single and large nucleus with centrally located, densely

staining nucleolus

Large endosome

Finely granulated cytoplasm

Large contractile vacuole

Exhibits small, spiny filaments for locomotion known as

acanthapodia

Moves sluggishly with polydirectional movement

Feed on gram-neegative bacteria, blue-green algae or

yeasts

Encysts when nutrients are depleted or environment is

Page 2 of 3

Free Living Pathogenic Amebae

unfavourable
Cannot be detected in CSF: only detected histologically
Multiplication and Life Cycle, Epidemiology

Progression into coma, death in weeks to months

Amoebic Keratitis

First diagnosed in 1974

Originally associated with the use of soft contact lenses

Organism isolated from solutions in lens cases

Viable trophozoites also adhere to lenses if these are not

properly cleansed and disinfected

Other predisposing factors: immunocompromised

patients

S/Sx
o Corneal lesions or ulceration
o Progressive corneal infiltration and clouding
o Iritis and scleritis
o Severe ocular pain
o Hypopyon and loss of vision

Treatment (will be further elaborated on treatment

section)
o Difficult, Corneal grafts are often required
Diagnosis

Found in soil, fresh and salt water

Reproduction
o Trophozoites are the reproductive stage
o Mode of Reproduction: Binary fission
Transmission
o Cyst is transmitted via dust or aerosols
o Trophozoite may also be infective (CDC)
Epidemiology
o Acanthamoeba have been isolated from
freshwater, seawater, ocean sediment, frozen
swimming water, distribution water, bottled
mineral water, industrial cooling water, air
conditioners, air, sewage, soil compost,
chlorinated swimming pools, medicinal pools,
dental treatment units and contact lenses.

Pathogenesis

Has three main points of entry:

o Eyes
o Nasal Passages
o Through breaks or ulcerations of the skin
Is able to spread hematogenously (thru blood)
Predisposing factor include: DM, alcoholism, pregnancy,
malnutrition, malignancy, immunosuppressive therapy,
surgical trauma, burns
Primary sites of infection include the skin or lungs. May
also infect the brain to cause GAE.
Clinical Manifestations

Granulomatous Amebic Encephalitis (GAE)

First discovered in 1972

Commonly occurs in immunosuppressed patients

Infection of the Brain:

o Most infected areas: Posterior fossa structures
such as Thalamus, Diencephalon and
Brainstem
o Affected areas have opaque leptomeninges with
purulent exudates and vascular congestion

S/Sx
o Non-specific - Fever, chills, fatigue and weight loss
o Common - Headache, confusion, somnolence,
coma, hallucinations and seizures
o Neurologic - Focal hemiparesis, cranial nerve
palsies, visual disturbances, ataxia, Increased
intracranial pressure which can cause
papilledema
o Other notable clinical manifestations Mental
abnormalities, meningism, localized signs and
coma
o Predominant
signs
and
symptoms
in
acanthamebic encephalitis are related directly to
the destructive encephalopathy and associated
meningeal irritation.

Incubation Period 10 days

Onset - Insidious with headache, personality changes,

slight fever
TRANSCRIBERS: PARAng Pag-ibig

Made only after death in majority of cases

Failure to diagnos is the result of:
o Initial lack of suspicion
o Inaccurate clinical diagnosis
Rapid progression of illness preventing completion of
diagnostic tests
Disease has high incidence in AIDS patients with low
CD4+ T-lymphovyte counts especially if it is associated
with skin lesions
Specific diagnosis depends on demonstrating the
trophozoites or cysts in tissues using histopathologic
stains and microscopy
Can also be detected by PCR using specific primer pairs
Acanthamoeba keratitis is diagnosed by epithelial biopsy
for histologic analysis and isolation of organism from
contact lens.
Etiologic agents include:
o A. castellani
o A. culbertsoni
o A. hutchetti
o A. polyhaga
o A. rhysoides
Treatment

For GAE

Appearance of cerebral manifestation: fatal outcome of

Acanthamebic encephalitis in 3-40 days

Some patients reported to respond with 5-fluorocytosine,

ketoconazole, itraconazole, pentamidine or amphotericin
B
For Amoebic Keratitis

Acanthamebic keratitis: previously most cases required

surgical excision of the infected cornea and corneal
transplantation

More recently: therapeutic keratoplaty not necessary if

multidrug treatment is started early (Cotromoxazole +
Pentamidine + Isethionate + Neosporin + Avoidance of
topical corticosteroids)

Advanced Acanthamebic keratitis surgery to remove

infected tissue ;Deep lamellar keratctomy with conjuntical
flap secured with interrupted 10-0 nylon sutures
Prevention and Control

No means of preventing exposure

Hygiene practices are important
Infection is common in lens wearers
o Proper and regular cleaning of lenses
Boiling water kills both cysts and trophozoites

Page 3 of 3