PARASITOLOGY LECTURE 10 – Intestinal Amebae, Commensal • life cycle is simple and no intermediate hosts involved

Amebae and Free-Lining Pathogenic Amebae • extracellularly located and do not undergo antigenic
Notes from Rivera,PT, Rivera,WL and Solon,JAA variation
USTMED ’07 Sec C – AsM • Cyst
o Quadrinucleate
INTESTINAL AMEBAE
o Resistant to gastic acidity and dessication
ENTAMOEBA HISTOLYTICA o Can survive in a moist environment for several
weeks
• Classification o Infection occurs when cysts are ingested from
o subphylum Sarcodina fecally-contaminated material
o superclass Rhizopoda o Modes of transmission
o class Lobosea  Fecal-oral route
o order Amoebida  Direct colonic inoculation through
o family Entamoebidae contaminated enema equipment
o Excystation occurs in the small or large bowel
o genus Entamoeba
a. nuclear fission
DISEASE
b. cytoplasmic division (forms 8
trophozoites)
• cause invasive intestinal and extraintestinal disease
immature cysts
MORPHOLOGY

• a pseudopod-forming nonflagellated protozoan parasite

• the most invasive in the Entamoeba family (which
includes E. dispar, E. hartmanni, E.polecki, E. coli and
E. gingivalis)
• only member of the family to cause colitis and liver
abscess mature cysts
• a eukaryotic organism
• cellular features
o lack organelles that resemble mitochondria
o no ER
o no Golgi apparatus
o cell surface and secreted proteins contain
signal sequences
o Ribosomes form aggregated crystalline arrays
in the cytoplasm in trophozoite
• biochemical characteristics
o lack glutathione metabolism
o uses pyrophosphate instead of ATP at several • Trophozoites
steps in glycolysis o Highly motile
o inability to synthesize purine nucleotides de o Possess pseudopodia
novo o Have the ability to colonize and/or invade the
o glucose is actively transported into cytoplasm large bowel (cysts are never found w/in
o end products of metab are EtOH and CO2 invaded tissues)
(acetate in aerobic conditions) o Multiply by binary fission
o Encyst
LIFE CYCLE a. produces uninucleate cysts
b. undergo 2 successive nuclear divisions
(forms quadrinucleate cysts)

PATHOGENESIS AND CLINICAL MANIESTATION

• most cases present as asymptomatic infections with

cysts being passed out in the stools (cyst carrier state)
• the non-pathogenic E. dispar has a higher prevalence
than E. histolytica
• most E. histolytica infections are asymptomatic in
endemic communities
• 2 stages • Amebic colitis
1. Infective cyst o Gradual onset of abdominal pain and diarrhea
2. invasive trophozoite form w/ or w/o blood and mucus in stools
o Fever occurs only in 1/3 of patients
• humans are the only known hosts o Intermittent diarrhea alternating with
 constipation this initial condition
o Children may develop fulminant colitis o Liver involvement occur through direct
 Severe bloody diarrhea extension from the intestinal ulcer
 Fever o In the liver, trophozoites lyse both
 Abdominal pain inflammatory and liver cells
o Abscess becomes filled with necrotic
proteinaceious debris (anchovy sauce-like
aspirate)
o Trophozoites are found at the edge of the
abscess

• Aspirate = odorless and bacteriologically sterile

(secondary bacterial invasion may occur)

colitis
• Ameboma
o Occurs in less than 1% of intestinal infections
o Mass-like lesion with abdominal pain and
history of dysentery
o Can be mistaken for carcinoma
• Amebic liver abscess (ALA)
o Most common extra-intestinal form of
amebiasis
o Cardinal manifestations (most frequent
complaints in acute cases {<2 weeks duration})
 Fever
 Right Upper Quadrant pain (RUQ) –
local or referred to the right
shoulder shoulder
o Liver is tender
o Hepatomegaly is present in 50% of cases
o Chronic disease (>2 weeks duration)
 found in older patients
 Wasting disease
 Weight loss rather than fever
o 72% of daily stool cultures harbored
trophozoites even in asymptomatic infections
o mortality uncomplicated <1%
• Complications
o Amebic Colitis:
 Perforation and Secondary Bacterial
peritonitis – most serious
complication
o ALA
 Rupture into the pericardium,
rupture into the pleura and super
infection – most serious
 Intraperitoneal rupture – second
most common complication (not as
serious because ALA is sterile)
o Secondary amebic meningoencephalitis –
considered in cases with abnormal mental
status
o Renal involvement caused by extension of ALA
ore retroperitoneal colonic perforation (rare)
o Genital involvement – caused by fistulae from
ALA and colitis or infection by sexual
transmission
• Immunity
o Natural or innate immunity
 In the intestines – involves mucin
inhibition of amebic attachment to
underlysing mucosal cells
 Systemic circulation – mechanism is
of complement-mediated killing of
trophozoites

o Acquired immunity
 Involves cell-mediated responses
and humoral responses
 Activated T-cells kills E. histolytica
i. by lysing trophozoites (by
contact)
PATHOLOGY ii. producing cytokines w/c
activate macrophages and
• E. histolytica named because of its ability to lyse human effector cells
iii. providing helper effect for
tissues
Bcell Ab production
• Invasive process initiated when trophozoite stage is able
to penetrate mucus layer covering the colonic o Amebic modulation of host immune responses
epithelium
 Infected subjects have been shown
• Invasion facilitated by expression of virulence factors
to be in a state of
1. Gal/Gal NAc lectin – mediates adherence to host immunosuppresion during the acute
cells stage
2. Amebapores – form pores in host cell membranes i. T cell hyporesponsiveness
3. cystein proteinases – cytopathic for host tissues ii. Depressed DTH
• In amebic invasion, Trophozoites… iii. Macrophage suppression
o Cause thinning of mucin layer action of
o Shortening of villi cystein DIFFERENTIAL DIAGNOSIS
o Breakdown of extracellular matrix proteases
• acute amebic colitis should be differentiated from
o Attach to mucosal cells facilitated by lectin bacillary dysentery of the ff. etiology: Shigella,
o Lyse cells amebapores Salmonella, Campylobacter, Yersinia, Entero-invasive E.
o Stimiulate release of IL8 which attracts and coli)
activates neutrophils o Fever and significantly elevated leukocyte
o Trophozoites erode the lamina propria and count are less common in amebic colitis
extend laterally producing characteristic flask-
shaped ulcer
Comparison of bacillary and Amebic dysentery
• Flask-shape ulcer = small defect in the mucosa and
larger area of necrosis in the submucosa and muscularis Baciliary Dysentery Amebic Dysentery
layers surrounded by normal epithelium Maybe epidemic Seldom epidemic
• Most common sites of amebic ulcer are cecum, Acute onset Gradual onset
ascending colon and sigmoid Prodromal fever and malaise No prodromal features
common
o From the primary site in the colon, Vomiting common No vomiting
trophozoites reach liver through portal vein Patient prostrate Patient usually ambulant
o Causes periportal inflammation Watery, bloody diarrhea Bloody diarrhea
 Amebic hepatitis – postulated for Odorless stool Fishy odor stool
Stool microscopy: numerous Stool microscopy: few bacilli,
bacilli, pus cells, macrophages, red cells, trophozoites with
red cells, no Charcot-Leyden ingested RBCs, Charcot-Leyden
crystals crystals
Abdominal cramps common and Mild abdominal cramps
severe TREATMENT AND PROGNOSIS
Tenesmus common Tenesmus uncommon
Natural history: spontaneous Natural history; lasts for • Treatment has 2 objections
recovery in a few days, weeks weeks; dysentery returns after 1. cure invasive disease at both intestinal and extra-
or more; no relapse remission; infection persists for intestinal sites
years 2. eliminate the passage of cysts from intestinal
lumen
• Differentiate amebic colitis from inflammatory bowel • Metronidazole is drug of choice
disease o Tinidazole and Secnidazole are also effective
o Amebic colitis should be ruled out before o Diloxanide furoate drug of choice for
steroid therapy is started because of risk of asymptomatic cyst passers.
developing toxic megacolon • Percutaneous drainage of live abscess
• ALA vs. pyogenic liver abscess, TB of the liver and o For patients who do not respond to
hepatic carcinoma metronidazole
• Genital amebiasis vs. carcinoma, TB, chancroid and o For prompt symptomatic relief of severe pain
lymphogranuloma venereum o For those with left lobe abscess that may
DIAGNOSIS rupture into the pericardium (and large and
multiple abscess with danger of rupture)
1. Microscopy
• standard method of diagnosis of trophozoites and cysts EPIDEMIOLOGY
in stool specimens

• for detection of trophozoites

o fresh stool sample examined w/in 30 minutes
from defecation
• use DFS with saline solution able to observe
trophozoite motility
o unidirectional movement is characteristic
• use saline and methyline blue
o Entamoeba species will stain blue
(differentiate from WBCs)
• Use saline and iodine
o Nucleus and karyosome can be observed
(differentiate E. histolytica from non-
pathogenic E. hartmanni, E. coli, Endolimax
nana) • infections occur worldwide but more prevalent in tropics
• Detection of E. histolytica trophozoite w/ ingested RBCs • worldwide cases 50 million, 100,000 of which end
is diagnostic of amebiasis. Charcot-Leyden crystals can fatally
also be seen in stool • second to malaria in terms of mortality caused by
protozoan parasites
• concentration methods such as Formalin Ether Conc Test • humans are major reservoirs of infection
(FECT) and Merthiolate Iodine Formalin Conc. Test • Mexico, Indonesia, Africa, Central and South America,
(MIFC) are more sensitive than DFS for detection of cysts Philippines
• morphologic structures observed: • Risk factors: treatment with corticosteroids, malignancy
1. size of cyst and malnutrition
2. number of nuclei
3. location and appearance of karyosome PREVENTION AND CONTROL
4. appearance of chromatoid bodies
5. presence of cytolasmic structures such as glycogen • improve environmental sanitation and sanitary disposal
vacuole of human feces, safe drinking water, safe food
• proper use of latrines and proper hygiene such as
• Species Identification of E. histolytica and E. dispar is washing hands
not possible for microscopy • Vaccines are cost-effective and a potent strategy for
o Done by PCR, ELISA and isoenzyme analysis amebiasis prevention

2. Serology
• Detection of antibodies in the serum is still the key COMMENSAL AMEBAE
diagnosis of ALA
• the presence of commensal amebae in stool is
• In ALA, microscopic detection cannot be done because
significant because:
aspiration is an invasive procedure and trophozoites are
missed because they are found at the periphery of the 1. may be mistaken for E. histolytica
abscess 2. indication of fecal contamination of food or water
• Serological tests: Indirect hemagglutination (IHAT),
MORPHOLOGY
counter immunoelectrophoresis (CIE), agar gel diffusion
(AGD), indirect fluorescent antibody test (IFAT) and
ELISA • differentiated from pathogenic E. histolytica
o IHAT can detect antibodies of past infection, • the three genera of intestinal amebae can be
even as long as 10 years differentiated through morphological features of their
• Abs demonstrated in asymptomatic infections so nuclei
serology can be used to monitor cyst carriers 1. Entamoeba –
o spherical nucleus
3. Radiographic o distinct nuclear membrane lined w/ chromatin
• Ultrasound, CT scan granules
and MRI – non-invasive o small karyosome near the center of the
and sensitive methods nucleus
in early detection of o trophozoites – usually only have one nucleus
ALA
2. Endolimax –
o vesicular nucleus
o large, irregularly-shaped karyosome anchored
 to nucleus by achromatic fibrils

3. Iodamoeba –
o large chromatin-rich karyosome surrounded by
a lyer of achromatic globules anchored to
nuclear membrane by achromatic fibrils

LIFE CYCLE

• all species have the following stages ENDOLIMAX NANA

1. Trophozoite
2. Precyst • small size of 6 to 15 um
3. Cyst • sluggish movement
4. Metacystic trophozoite • characteristic Endolimax nucleus (w/ large irregular
o exception of E. gingivalis w/c has no cyst karyosome)
stage and does not inhabit the intestines • cysts are quadrinucleate when mature
• cysts pass through the acidic stomach unscathed,
cyst
protected by cyst walls
• excystion occurs in the alkaline environment of lower
small intestines
• Metacystic trophozoites colonize large intestines and
live on mucus coat covering intestinal mucosa
• Amebae are non-invasive and do not cause disease
• Reproduction by binary fission of trophozoites
• Encystion occurs as amebae pass through lower colon
trophozoite

ENTAMOEBA HARTMANNI

• similar to E. histolytica except that it is much smaller

and does not ingest RBCs
• more sluggish movement
• mature cyst are quadrinucleated and have a coarse
cytoplasm IODAMOEBA BüTSCHLII
• immature cysts have chromatoidal bars (short with
tapered ends, or thin and bar-like) • trophozoite is usually 9-14 um long
• characteristic large vesicular nucleus w/ large endosome
cyst surrounded by achromatic granules
• no peripheral chromatin granules on nuclear membrane
• cysts is uninucleated w/ large glycogen body w/c stains
deeply w/ iodine

cyst

trophozoite

trophozoite

ENTAMOEBA COLI

• cosmopolitan distribution
• harmless inhabitant of colon
• typical Entamoeba nucleus
• Trophozoite can be diff. from E. histolytica by features:
DIAGNOSIS
o More vacuolated or granular ER w/ bacteria
and debris but no RBCs
• stool examination
o Narrower, less differentiated ectoplasm
• liquid stools will show trophozoites
o Broader, blunter pseudopodia • formed stools will show cysts
o More sluggish undirected movements
• DFS to demonstrate trophozoites
o Thicker, irregular peripheral chromatin w/
• Formalin-ether concentration technique to differentiate
large eccentric karyosomes in the nucleus
species
• Cysts of E. coli vs. E. histolytica
o Larger size TREATMENT
o Greater number of nuclei (8 vs 4 in E.
histolytica) • not necessary because does not cause disease
o More granular cytoplasm
o Splinter-like chromatoidal bodies EPIDEMIOLOGY

cyst • human infection by ingestion of viable cysts in food or

water
• 21% of Filipinos infected with E.coli, 9% - Endolimax
nana, 1% Iodamoeba butschlii

PREVENTION AND CONTROL

• The usual…
trohpozoites
FREE-LIVING PATHOGENIC AMEBAE  Chills
 Fatigue
LIFE CYCLE
 Weight losss
o Common
 Headache
 Confusion
 Somnolence
 Coma
 Hallucinations
 Seizures
o Neurologic symptoms
 Focal hemiparesis
 Cranial nerve palsies
 Visual disturbances
 Ataxia
o Increased intracranial pressure can cause
papilledema
o Skin lesions are an important diagnostic
feature of the infection
• Incubation period is about 10 days
o w/ subacute and chronic clinical course of
infection that lasts for several weeks
o clinical manifestations
 mental abnormalities
 meningism
 localized neurological signs
 coma
• cerebral hemispheres
o edematous
o soft with hemorrhages and abscesses
o most affected areas are posterior fossa,
diencephalons, thalamus and brainstem
o leptomeninges are opaque w/ purulent
exudates
ACANTHAMOEBA

• route of invasion and penetration into the CNS via the

circulatory system
• primary sites of infection are skin or lungs • also an ocular surface pathogen that causes amebic
keratitis
DISEASE o associated with the use of soft contact lenses
o viable trophozoites can adhere to lenses if not
• causes granulomatous amebic encephalitis (GAE) properly cleaned and disinfected
o signs and symptoms
MORPHOLOGY  corneal ulceration
 progressive corneal infiltration and
• Trophozoites clouding
o trophozoites exhibit a single and large nucleus  iritis
with a centrally-located, densely staining  scleritis
nucleolus, a large endosome, finely granulated  s
cytoplasm and a large contractile vacuole eve
o exhibit small, spiny filaments for locomotion re
known as acanthapodia
o sluggish movement w/ polydirectional
movement
• Cysts
o Double-walled w/ outer wrinkeled wall and an
inner polygonally-shaped wall
o Pores or ostioles are seen at the point of pain
contact bet the two walls  hypopyon
• Presence of naturally-occuring bacterial endosymbionts  loss of vision
in Acanthamoeaba sp. o often confused w/ fungal or herpetic keratitis

LIFE CYCLE Amebic keratitis

• a small free-living ameba 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
• Encysts if the environment is not favorable
• A ubiquitous organism
PATHOGENESIS AND CLINICAL MANIFESTATION
• causes GAE
o occur in chronically ill and debilitated
individuals
o with impaired immune defense mechanisms
o under immunosuppressive therapy
• Signs and symptoms of GAE are related to destructive
encephalopathy and associated w/ meningeal irritation
o Non-specific constitutional manifestations
 Fever
DIAGNOSIS
• GAE (A. encephalitis)
o Made only after death in majority of cases
o Failure to diagnose is the result of a variety of
factors
 Lack of initial suspicion
 Inaccurate early clinical diagnosis
 Rapid progression of illness
o Has high incidence in AIDS patients w/ low
CD4+ T-lymphocytes counts, especially if
associated w/ skin lesions and/or sinusitis
o specific diagnosis
  demonstration of trophozoites or o Allows it to move towards a food souce more
cysts in tissues by microscopy rapidly
 can be isolated from CSF and
cultured Trophozoite in spinal fluid Trophozoite in CSF
• A. keratitis
o Epithelial biopsy for histologic analysis
o Isolation of organisms from lens of contact
lens weares
o Etiologic agents:
 A. castellani
 A. culbertsoni
 A. hutchetti
 A. polyphaga
 A. rhysoides LIFE CYCLE

TREATMENT AND MANAGEMENT

• trophozoite stage that can transform reversibly into
non-reproductive flagellate or a resistant cyst
• w/ appearance of cerebral manifestation, A. • transformation can take place w/in a period of 2-3 hours
encephalitis has a fatal outcome in 3-40 days or up to 3-4 days
o some patients respond to treatment with:
 5-fluorocytosine PATHOGENESIS AND CLINICAL MANIFESTATION
 ketoconazole
 itraconazole • causes gastritis and diarrhea
 pentamidine • causes primary amebic meningoencephalitis (PAM) – rare
 amphotericin B disease that leads to inflammation of the brain and
• A. keratitis treatment achieved w/ multidrug treatment destruction of brain tissue
if started early • N. fowleri able to survie in elevated temperatures
o Clotrimazole + (46oC) and 0.5 ug/ml of hyperchlorinated water
o Pentamidine, • PAM is characterized by fever, headache, vomiting, signs
o Isethionate, of meningeal irritation and encephalitis with rapid
o Neosporin progression to coma and death
o – avoidance of corticosteroids o CSF findings of pleocytosis
o surgery for A. keratitis o High percentage of polymorphonuclear cells
o Hypoglycorrhachia
EPIDEMIOLOGY o Eleveated protein leves

• Acanthamoeba species have been isolated from all kinds
of water (sea, fresh, mineral,etc), air, sewage, soil,
compost, vegetables, mushrooms, fish, reptiles, birds,
mammals
• in humans, isolated in nasal cavity, throat and intestines
as well as cerebral tissue, lung tissue, skin wounds and
cornea
• encephalitis in US
• Keratitis in Japan, Korea, South America, Germany
PREVENTION AND CONTROL
• boiling water is the best possible way of killing
trophozoites and cysts
• regular disinfection of contact lenses
NAEGLERIA
• free-living amebo-flagellate
• can exists as an ameba (trophozoite form) and as a
flagellate (swimming form)
• Naegleria gruberi most commonly studied non-
pathogenic species
• Pathogenic species, N. fowleri, causes fatal
meningoencephalitis in humans and laboratory animals
such as mice
o Causes degenerative or cytopathic effects in
cell cultures
• Non-pathogenic can be distinguished from pathogenic by
a combinations of cell morphology, culture medium
preference, temperature tolerance, lectin sensitivity,
isozyme pattern, DNA restriction patterns, mouse
pathogenicity and serology
• Locally occurring species named N. philippinensis
• Mode of transmission – oral or intranasal routes while
swimming in contaminated pools, lakes and rivers
MORPHOLOGY
DIAGNOSIS
• diagnosis of PAM – based on presence of trophozoites in
brain and CSF
• Naegleria trophozoites can be identified by presence of
blunt, lobose pseudopodia and directional motility
TREATMENT
• Amphotericin B – drug of choice for treating PAM
o Induces changes in nucleus and mitochondria
o Increases proliferation of both rough and
smooth ER
o Decreases number of food vacuoles
o Increases formation of autophagic vacuoles
o Inhibiting pseudopod formation
o Induces blebbing of the ameba plasma
membrane
• N. fowleri
o tolerant between 65o - 100o C
o inhibted by 0.2 NaCl and KCl
o CaCl2 stimulates encystment
o Drying is lethal to trophozoites
o Cysts remain viable if rehydrated w/in 23
months
o Cysts are non viable if lyophilized
EPIDEMIOLOGY
• all types of water
• soil is preferred habitat
PREVENTION AND CONTROL
• avoid diving into and swimming gin warm and stagnant
freshwater pools, discharge pools, unchlorinated poorly
maintained poos or mud-lined lakes and ponds

• Trophozoite easily recognizable under phase contrast

microscope
o Characteristic lobose monopseudopodium
o Very prominent nucleus w/ centrally-located
nucleolus
o Forms a pair of flagella originating from the
tip of a pear-shaped cell body
o Transforms into a biflagellated organism
• Flagellated organism
-fin-

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