The Amoebas

• With one exception, there are two morphologic forms

CHAPTER OUTLINE in the amebic life cycle.
I. Protozoa ▪ Trophozoites
→ the form that feeds, multiplies, and
II. Morphology and Life Cycle
possesses pseudopods
i. Trophozoites
ii. Cysts ▪ Cysts
→ the nonfeeding stage characterized by a
III. Laboratory Diagnosis thick protective cell wall designed to
protect the parasite from the harsh outside
IV. Pathogenesis and Clinical Symptoms
environment when deemed necessary
V. The Amoeba Classification
• The nuclear characteristics of trophozoites are
VI. Intestinal Species basically identical to those of their corresponding
i. Entamoeba histolytica
cysts.
ii. Entamoeba hartmanni
iii. Entamoeba coli
iv. Entamoeba polecki
v. Endolimax nana TROPHOZOITES
vi. Iodamoeba bütschlii
• Trophozoites are characteristically delicate and
VII. Extraintestinal Species
fragile and, because of their ability to produce and
i. Entamoeba gingivalis
use pseudopods, motile.
ii. Naegleria fowleri
iii. Acanthamoeba spp.
• The life cycles of all the intestinal amebas are similar.

PROTOZOA • The most common means whereby amebas are

transferred to humans is through ingestion of the
• Protozoa
infective cyst in contaminated food or water.
→ unicellular organisms and the lowest form of ▪ In most cases, trophozoites are easily
animal life destroyed by the gastric juices of the stomach.

• In the subkingdom Protozoa, there are three phyla of • Trophozoites are also susceptible to the environment
medical interest in humans. outside the host. Therefore, trophozoites are not
▪ The phylum Sarcomastigophora, subphylum usually transmitted to humans.
Sarcodina, includes the pathogenic and
nonpathogenic amebas.
• Excystation
→ the morphologic conversion from the cyst form
into the trophozoite form
MORPHOLOGY AND LIFE CYCLE
→ occurs in the ileocecal area of the intestine
• The most important feature that separates amebas
from the other groups of unicellular Protozoa is the • Replication only occurs in the trophozoite stage; it is
means by which they move. accomplished by multiplication of the nucleus via
asexual binary fission.
• Amebas are equipped with the ability to extend their
cytoplasm in the form of pseudopods (often referred
to as false feet), which allows them to move within
their environment.

47 PADAYON, FUTURE RMT, MD! | BS Public Health – Batch LEVIS

 CHAPTER 04: The Amoebas PH 201
SECOND YEAR – SECOND TERM | Para

CYSTS • The appearance of key nuclear characteristics, such

as the number of nuclei present and the positioning
• Encystation of the nuclear structures, is crucial to differentiate
→ the conversion of trophozoites to cysts the amebas correctly.
→ occurs in the intestine when the environment ▪ The presence of other amebic structures and
becomes unacceptable for continued characteristics, such as cytoplasmic
trophozoite multiplication inclusions and motility, also aids in the
identification of the amebas.
• A number of conditions individually or in
combination may trigger encystation. • Standard microscopic procedures include
▪ ameba overpopulation examination of specimens for amebas using saline
▪ pH change wet preparations, iodine wet preparations, and
▪ food supply (too much or too little) permanent stains.
▪ available oxygen (too much or too little)
• The saline wet preparation and iodine wet
• Contrary to the trophozoites, cysts are equipped with preparation each have an advantage that supports
a protective cell wall. their use.
▪ The cell wall allows cysts to enter the outside ▪ Saline wet preparations are of value because
environment with the passage of feces and they will often show motility of the amebic
remain viable for long periods of time. trophozoites.

• The ingestion of the infective cysts completes the • The internal cytoplasmic, as well as the nuclear
typical intestinal amebic life cycle. structures, may be more readily seen with the use of
iodine wet preparations.
• Most cyst-forming amebae go through nuclear
division, and then divide again after excystation in a • It is important to note that permanent smear
new host. procedures of samples suspected of having amebas
must be performed to confirm parasite
identification.
LABORATORY DIAGNOSIS ▪ In most cases, the key identifying
characteristics cannot be accurately
• Amebic trophozoites as well as cysts may be seen in
distinguished without the permanent stain.
stool samples submitted for parasite study.
▪ The permanent stain allows for many of the
otherwise refractive and invisible structures to
• Trophozoites are primarily recovered from stools
be more clearly visible and thus easier to
that are of soft, liquid, or loose consistency.
identify.
▪ The permanent smear procedure may,
• Formed stool specimens are more likely to contain
however, shrink amebic parasites, causing
cysts.
measurements smaller than those typically
seen in wet preparations.
• The morphologic forms present in samples other
than stool are noted on an individual basis. It is
important to point out that the presence of either or
both morphologic forms is diagnostic.

• Proper determination of organism size, using the

ocular micrometer, is essential when identifying the
amebas.

48 PADAYON, FUTURE RMT, MD! | BS Public Health – Batch LEVIS

 CHAPTER 04: The Amoebas PH 201
SECOND YEAR – SECOND TERM | Para

PATHOGENESIS AND THE AMOEBA CLASSIFCATION

CLINICAL SYMPTOMS
• The amebas, members of the subphylum Sarcodina
• A number of patients infected with intestinal amebas and class Lobosea, may be separated into two
are asymptomatic. categories, intestinal and extraintestinal (meaning
• Amebas are often discovered, however, in patients parasites that migrate and/or take up residence
suffering from diarrhea without an apparent cause. outside the intestines).

• Diagnosis of nonpathogenic amebas is important

because this finding suggests that the ingestion of
contaminated food or drink may have occurred.

• The presence of pathogenic amebas in addition to

nonpathogenic amebas is also possible because the
transmission route of both amebic groups is
identical.

• Thorough screening of such samples is crucial to AMOEBAS

ensure proper identification of all parasites that may Subphylum Mastigophora
be present. Class Zoomastigophora
INTESTINAL SPECIES
• Infection is most common in people who live in Entamoeba histolytica
underdeveloped countries that have poor sanitary Entamoeba hartmanni
Entamoeba coli
conditions.
Entamoeba polecki
▪ In the United States, amebiasis is often found
Endolimax nana
in immigrants from and people who have
Indamoeba bütschlii
traveled to underdeveloped countries. EXTRAINTESTINAL SPECIES
Entamoeba gingivalis
• Furthermore, amebas may be present and thus Naegleria fowleri
infect individuals in areas and institutions in which Acanthamoeba spp.
crowded conditions prevail.

• It is important to note that only one of the intestinal

amebas, E. histolytica, may produce characteristic
symptoms, and is universally considered to be a
pathogen.

• Infections with each of the extraintestinal amebas

may cause symptoms that are other than intestinal
in nature, often involving such areas as the mouth,
eye, and brain.

49 PADAYON, FUTURE RMT, MD! | BS Public Health – Batch LEVIS

 ENTAMOEBA HISTOLYTICA
MUST KNOW

→ causes intestinal amebiasis, amebic colitis, amebic

dysentery, extraintestinal amebiasis
→ currently classified within the subphylum Sarcodina,
superclass Rhizopoda, class Lobosea, order Amoebida,
family Entamoebidae, and genus Entamoeba
→ characterized by having a vesicular nucleus, a centrally
(or near central) located small karyosome, and varying
numbers of chromatin granules adhering to the nuclear
membrane
→ parasite names are often shortened to just the first letter
of the genus followed by the species name; E. histolytica
is the abbreviated version of Entamoeba histolytica
→ a pseudopod-forming non-flagellated protozoan
parasite; the most invasive of the Entamoeba parasites
→ Diagnostic Stage: quadrinucleated cyst
→ Infective Stage: cyst

MORPHOLOGY
Trophozoite Cyst
Size Range 8-65 µm Size Range 8-22 µm

Motility progressive, finger-like Shape spherical to round

pseudopodia
Number of Nuclei one to four
Number of Nuclei one
Karyosome small and central
Karyosome small and central
Peripheral Chromatin fine and evenly distributed
Peripheral Chromatin fine and evenly distributed
Cytoplasm finely granular
Cytoplasm finely granular (having a
ground glass in appearance) Cytoplasmic Inclusions chromatoid bars (rod-shaped
Cytoplasmic Inclusions ingested RBCs or cigar-shaped), rounded
ends in young cysts
• The trophozoite are highly motile which exhibits rapid, diffuse glycogen mass in
unidirectional, progressive movement, achieved with the young cysts
help of finger-like hyaline pseudopods.
• The presence of a highly refractile hyaline cyst wall
• Variants of the karyosome include eccentric or helps in the recognition of this morphologic form.
fragmented karyosomal material.
• Young cysts characteristically contain unorganized
• Although the karyosome and peripheral chromatin chromatin material that transforms into squared or
appearance may vary, most trophozoites maintain the round-ended structures call chromatoid bars, defined
more typical features described. as structures that contain condensed RNA material.
• The invisible nucleus in unstained preparations becomes • A diffuse glycogen mass, a cytoplasmic area without
apparent when stained. defined borders that is believed to represent stored
▪ Stained preparations may reveal lightly staining fibrils food, is also usually visible in young cysts. As the cyst
located between the karyosome and peripheral matures, the glycogen mass usually disappears, a
chromatin. process that likely represents usage of the stored food.
• Bacteria, yeast, and other debris may also reside in the • The mature infective cyst is quadrinucleated
cytoplasm, but their presence, however, is not diagnostic. (containing four nuclei).
• The hyaline pseudopodium is formed when the clear, • RBCs, bacteria, yeast, and other debris are not found in
glasslike ectoplasm, or outer layer is extruded, and the the cyst stage.
granular endoplasm flows into it. Ingested red blood cells
are observed as pale, greenish, refractile bodies in the
cytoplasm of the ameba.

50
 ENTAMOEBA HISTOLYTICA
LIFE CYCLE
Notes
• Mode of Transmission: ingestion of
fecally-contaminated material;
venereal transmission through fecal-
oral contact or direct colonic
inoculation through contaminated
enema equipment
• Diagnostic Stage: quadrinucleated
cyst
• Infective Stage: cysts
• The life cycle of E. histolytica consists
of two stages: an infective cyst and an
invasive trophozoite form.
• No host other than humans is
implicated in the life cycle, although
natural infection of primates has been
reported.
• Once the infective cyst is ingested,
excystation occurs in the small
intestine.
• As a result of the nuclear division, a
single cyst produces eight motile
trophozoites. These motile amebas
settle in the lumen of the large
intestine, where they replicate by
binary fission and feed on living host
cells.
• On occasion, trophozoites migrate to
other organs in the body, such as the
liver, and may cause abscess
formation. Unless these trophozoites
return to the lumen of the large
intestine, their life cycle ceases and
diagnosis in such cases will rely on
serologic testing.
• Encystation occurs in the intestinal
lumen, and cyst formation is
Cycle complete when four nuclei are
present.
Diagnostic Stage
• These infective cysts are passed out
1. Cysts and trophozoites are passed in feces. Cysts are typically found in into the environment in human feces
formed stool, whereas trophozoites are typically found in diarrheal stool. and are resistant to a variety of
Infective Stage physical conditions.
2. Infection with Entamoeba histolytica (and E. dispar) occurs via ingestion of • The quadrinucleated cyst is resistant
mature cysts image from fecally contaminated food, water, or hands. to gastric acidity and desiccation, and
Exposure to infectious cysts and trophozoites in fecal matter during sexual can survive in a moist environment for
contact may also occur. several weeks.
Excystation • Survival in a feces-contaminated
3. Excystation occurs in the small intestine and trophozoites are released, environment for up to 1 month is
which migrate to the large intestine. common.
4. Trophozoites may remain confined to the intestinal lumen. • It is important to note that in addition
• Noninvasive Infection: with individuals continuing to pass cysts in their to cysts, trophozoites, under the right
stool (asymptomatic carriers) conditions, may also be present in the
• Intestinal Disease: trophozoites can invade the intestinal mucosa stool. Liquid or semi-formed samples
• Extraintestinal Disease: blood vessels, reaching extraintestinal sites such may show trophozoites if the intestinal
as the liver, brain, and lungs motility is rapid.
Binary Fission • Cysts will form, on the other hand, if
the intestinal motility is normal.
5. Trophozoites multiply by binary fission and produce cysts, and both stages
are passed in the feces.
Diagnostic Stage
6. Cysts can survive days to weeks in the external environment and remain
infectious in the environment due to the protection conferred by their walls.
Trophozoites passed in the stool are rapidly destroyed once outside the
body, and if ingested would not survive exposure to the gastric
environment.

51
 ENTAMOEBA HISTOLYTICA
CLINICAL SYMPTOMS LABORATORY DIAGNOSIS
• Entamoeba histolytica is the only known pathogenic intestinal ameba. Specimen of Choice
• The proposed mechanisms for virulence are production of enzymes or other
→ stool
cytotoxic substances, contact-dependent cell killing, and cytophagocytosis.
→ material collected from a
▪ In vitro, amebic killing of target cultivated mammalian cells involve receptor-
sigmoidoscopy procedure
mediated adherence of ameba to target cells, amebic cytolysis of target
→ hepatic abscess material
cells, and amebic phagocytosis of killed or viable target cells. E. histolytica
trophozoites adhere to the colonic mucosa through a galactose-inhibitable • The diagnosis of E. histolytica
adherence lectin (Gal lectin). infection may be accomplished
▪ Then, the amebae kill mucosal cells by activation of their caspase-3, leading by standard and alternative
to their apoptotic death engulfment. methods.
• Recent studies have shown that susceptibility of humans to E. histolytica • In addition to performing
infection is associated with specific alleles of the HLA complex. traditional wet preparation and
• Ameboma occurs in less than 1% of intestinal infections. It clinically presents as a permanent staining techniques on
a suspected stool sample,
mass-like lesion with abdominal pain and a history of dysentery. It can be
material collected from a
mistaken for carcinoma. Asymptomatic ameboma may also occur.
sigmoidoscopy procedure, as well
• Amebic Liver Abcess (ALA) as hepatic abscess material, may
→ the most common extra-intestinal form of amebiasis be processed and examined in the
→ The cardinal manifestations of ALA are fever and right upper quadrant (RUQ) same manner.
pain. • TYI-S-33: a special medium that
→ The onset of amebic colitis may be sudden after an incubation period of 8 to supports E. histolytica in culture
10 days, or after a long period of asymptomatic cyst carrier state. • When E. histolytica is suspected
• The range of symptoms varies and depends on two major factors. but not recovered in stool
▪ the location(s) of the parasite in the host samples, other laboratory tests,
▪ the extent of tissue invasion including immunologically based
procedures, may be used.
• Asymptomatic Carrier State
→ Three factors, acting separately or in combination, are responsible for the • Charcot-Leyden crystals can also
asymptomatic carrier state of a patient infected with E. histolytica. be seen in the stool.
▪ the parasite is a low-virulence strain • Using saline and methylene blue,
▪ the inoculation into the host is low Entamoeba species will stain blue,
▪ the patient’s immune system is intact thus, differentiating them from
white blood cells.
→ In these cases, amebas may reproduce but the infected patient shows no
clinical symptoms. • Stool culture using Robinson’s and
Inoki medium is more sensitive
• Symptomatic Intestinal Amebiasis than stool microscopy but is not
▪ Amebic Colitis routinely available.
→ an intestinal infection caused by the presence of amebas exhibiting
• Ultrasound, computerized
symptoms tomography (CT scan), and
→ these patients may transition from amebic colitis into amebic dysentery magnetic resonance imaging
(a condition characterized by blood and mucus in the stool), in some (MRI) are non-invasive and
cases sensitive methods in early
→ may exhibit non-descript abdominal symptoms or may complain of more detection of ALA.
specific symptoms, including diarrhea, abdominal pain and cramping, • Methods Currently Available
chronic weight loss, anorexia, chronic fatigue, and flatulence → antigen tests
▪ Secondary bacterial infections may develop after the formation of flask- → enzyme-linked
shaped amebic ulcers in the colon, cecum, appendix, or rectosigmoid area of immunosorbent assay (ELISA)
the intestine. → indirect hemagglutination
(IHA)
▪ As noted, stools recovered from patients suffering from amebic dysentery are → gel diffusion precipitin (GDP)
characterized by the presence of blood and/or pus and mucus. → indirect immunofluorescence
• Symptomatic Extraintestinal Amebiasis (IIF).
▪ E. histolytica trophozoites that migrate into the bloodstream are removed by • Serologic tests designed to detect
and take up residence in the liver. E. histolytica are available and are
→ The formation of an abscess in the right lobe of the liver and trophozoite typically only helpful in cases of
extension through the diaphragm, causing amebic pneumonitis, may extraintestinal infections.
occur.
→ Patients in this state often exhibit symptoms similar to those of a liver TREATMENT
infection plus a cough, with the most common of the symptoms being
upper right abdominal pain with fever. • Treatment regimens for patients
→ Weakness, weight loss, sweating, pronounced nausea, and vomiting may infected with E. histolytica vary by
occur, as well as marked constipation with or without alternating diarrhea. the type of infection present.

▪ In addition to the liver, E. histolytica has been known to migrate to and infect • Asymptomatic: paromomycin,
diloxanide furoate (Furamide), or
other organs, including the lung, pericardium, spleen, skin, and brain.
metronidazole (Flagyl)
▪ Venereal amebiasis may also occur.
• Asymptomatic Intestinal
▪ Men become infected with penile amebiasis after experiencing unprotected Amebiasis: iodoquinol,
sex with a woman who has vaginal amebiasis. paromomycin, or diloxanide
▪ The disease may also be transferred during anal intercourse. furoate
• Symptomatic Intestinal Amebiasis:
▪ It is interesting to note that on examination of these genital areas, the
metronidazole or tinidazole
trophozoite form of E. histolytica is most commonly encountered.

52
 ENTAMOEBA HISTOLYTICA
EPIDEMIOLOGY
Who?
→ immigrants
→ travelers from endemic countries
→ homosexual males (men having sex with men)
→ HIV patients
→ institutionalized people
Where?
→ subtropical and tropical areas of the world, also in colder
climates
→ places at which human waste is used as fertilizer
→ areas of poor sanitation
→ hospitals for the mentally ill
→ prisons
→ day care centers
→ prevalent in the Indian subcontinent, Africa, East Asia, and South
and Central America
How?
→ Ingestion of food and drink contaminated with E. histolytica
cysts from human feces
→ direct fecal-oral contact
→ unprotected sex
→ Flies and cockroaches may also serve as vectors (living carriers
responsible for transmitting parasites from infected hosts
uninfected hosts) of E. histolytica by depositing infective cysts
on unprotected food.
→ improperly treated water supplies
Notes
• Humans: the major reservoirs of infection with E. histolytica
• For a long time, the species-complex referred to as E. histolytica
was believed to infect 500 million people, or 10% of the world’s
population.
• However, with the recent redescription into three different species:
the pathogenic E. histolytica, and the commensals, E. dispar and E.
moshkovskii, the true prevalence of amebiasis is approximately 1
to 5% worldwide.
• There are 50 million E. histolytica infection cases, and 40,000 to
100,000 deaths due to amebiasis in the world per year.
• Amebiasis is the third most important parasitic disease, after
malaria and schistosomiasis, and second to malaria as the top
cause of mortality among parasitic protozoans.
• Entamoeba histolytica infection occurs in as many as 10% of the
world’s population and is considered a leading cause of parasitic
deaths after only malaria, the clinical manifestation of infection
with Plasmodium species parasites, and schistosomiasis, the
umbrella term for the disease associated with Schistosoma spp.
infection.
• In developing countries, prevalence depends on the level of
sanitation, crowding, socio-economic status, cultural habits, and
age.
• In developed countries, infection is usually caused by E. dispar,
and is prevalent in certain groups.
PREVENTION & CONTROL
Individual Level
✓ uncontaminated water: by boiling or treating with iodine crystals
✓ properly washing food products
✓ avoiding the use of human feces as fertilizer
✓ good personal hygiene and sanitation practices
✓ protection of food from flies and cockroaches
✓ avoidance of unprotected sexual practices
Community Level
✓ safe water supply: water treatment regimen that includes
filtration and chemical treatment
✓ proper public sanitation practices
✓ health education and promotion
✓ vaccination
NOTES OF INTEREST
• The members of the genus Entamoeba are
characterized by having a vesicular nucleus, a
centrally (or near central) located small
karyosome, and varying numbers of chromatin
granules adhering to the nuclear membrane.
• These nuclear and other morphologic
differences distinguish the species of
Entamoeba except E. histolytica, E. dispar, and
E. moshkovskii (previously known as the Laredo
strain).
• The three said species are morphologically
identical and of the same size.
▪ E. hartmanni, formerly referred to as “small
race” of E. histolytica, is differentiated
primarily on the basis of size.
• Several discoveries during the late 1880s led to
the confirmation that E. histolytica was indeed
a pathogen.
▪ Of particular note was the work of Loesch,
who studied the stool of a patient suffering
from dysentery. The ameba-infected stool
from this patient was transferred to a dog
for further study.
• The overall prevalence of Entamoeba infection
in the United States is approximately 4%.
Entamoeba spp. infect approximately 10% of
the world’s population.
• The prevalence of infection is as high as 50% in
areas of Central and South America, Africa,
and Asia.
• Of all of the cases of E. histolytica worldwide,
only 10% progress to the invasive stage.
• Invasive and noninvasive strains of E.
histolytica may be distinguished by performing
isoenzyme electrophoresis and examining the
zymodemes (isoenzyme patterns). These
analyses are conducted primarily for
epidemiologic studies of the organism.
Applications of isoenzyme electrophoresis are
not, however, useful in routine laboratory
testing.
• The World Health Organization (WHO)
recommends that intestinal infection be
diagnosed with an E. histolytica–specific test,
thus rendering the classic stool ova and
parasite examination obsolete in this setting.
• However, finding quadrinucleated cysts or
trophozoites containing ingested erythrocytes
in stool is considered by many to be diagnostic
for amebic colitis.
• A nonpathogenic ameba, known as
Entamoeba dispar, has been identified that is
morphologically identical to E. histolytica.
Thus, it is often impossible to distinguish
▪
these two ameba based on morphology
alone.
▪ Because of this inability to distinguish these
two like parasites, the laboratory often
reports both names if trophozoites that lack
RBCs and/or cysts are recovered.
▪ If, however, trophozoites are seen that
contain ingested RBCs, it is then
appropriate to report them as E. histolytica.
▪ In cases for which identification is not
apparent, speciation requires specialized
testing methodologies that include DNA
probes and electrophoresis techniques
designed to target enzymes.

53
 ENTAMOEBA HARTMANNI
MUST KNOW
→ causes intestinal amebiasis, amebic colitis, amebic
dysentery, extraintestinal amebiasis.
→ The appearance of E. hartmanni is relatively similar to
that of E. histolytica apart from its smaller size.
→ Diagnostic Stage: cyst, trophozoite
→ Infective Stage: cyst

MORPHOLOGY
Cyst
Trophozoite
Size Range 5-15 µm

Motility nonprogressive, finger-like

pseudopods

Number of Nuclei one

Karyosome small and central

Peripheral Chromatin fine and evenly distributed

(beaded appearance)

Cytoplasm finely granular

Trophozoite

Cytoplasmic Inclusions ingested bacteria may be

present
LABORATORY DIAGNOSIS
Specimen of Choice stool
• Trophozoites contain one nucleus that is not typically
visible in unstained preparations. • It is important to note that the size ranges of E. histolytica
• Nuclear structure variations identical to those described and E. hartmanni overlap. Specific identification based
in the discussion of E. histolytica may occur. only on size in such cases is impossible.
• Unlike that of E. histolytica, the cytoplasm of E. • The protocol for reporting such findings varies by
hartmanni does not contain ingested red blood cells. laboratory.
• Proper use of the ocular micrometer is therefore essential
Cyst to obtain correct measurements of suspected organisms.
Size Range 5-12 µm
CLINICAL SYMPTOMS
Shape spherical
• Infections with E. hartmanni are typically asymptomatic.
Number of Nuclei one to four
TREATMENT
Karyosome small and central
• Although some questions exist regarding the
Peripheral Chromatin fine and evenly distributed pathogenicity of E. hartmanni, it is generally considered a
nonpathogen and treatment is usually not indicated.
Cytoplasm finely granular
EPIDEMIOLOGY
Cytoplasmic Inclusions chromatoid bars, rounded
ends in young cysts Who?
diffuse glycogen mass in → people who have traveled to tropical places that have
young cyst poor sanitary conditions
→ immigrants from tropical countries that have poor
• Nuclei located in the E. hartmanni cysts are sanitary conditions
characteristically smaller in size than the single nucleus → people who live in institutions that have poor sanitary
of its corresponding trophozoite. conditions
• The cysts of E. hartmanni develop just as those of E. → men who have sex with men
histolytica. Where?
• Mature cysts are quadrinucleated like E. histolytica, and → cosmopolitan areas
have rod-shaped chromatoid material with rounded or → areas with poor sanitation and hygiene practices
squared ends. How?
→ ingestion of infected cysts present in contaminated
NOTES OF INTEREST food or water
• The means whereby food and water become PREVENTION & CONTROL
contaminated are similar to those of E. histolytica.
• Entamoeba hartmanni was at one time designated as Individual Level
“small race” E. histolytica because of the many ✓ exercising proper personal hygiene
similarities between the two organisms. ✓ protection of food from flies and cockroaches
• Serologic differences between E. hartmanni and E. Community Level
histolytica have been described. ✓ proper public sanitation practices

54
 ENTAMOEBA COLI
MUST KNOW
→ causes intestinal amebiasis, amebic colitis, amebic
dysentery, extraintestinal amebiasis.
→ Diagnostic Stage: cyst, trophozoite
→ Infective Stage: cyst

MORPHOLOGY
Trophozoite
Size Range 12-55 µm

Motility
nonprogressive, blunt pseudopods

Number of Nuclei one

Karyosome large, irregular shape, eccentric

Peripheral Chromatin
unevenly distributed
LABORATORY DIAGNOSIS
Cytoplasm coarse and granulated
Specimen of Choice stool
Cytoplasmic vacuoles containing bacteria often
• Although not considered as being pathogenic, the
Inclusions visible
presence of E. coli suggests ingestion of
• It can be differentiated from E. histolytica trophozoite by the contaminated food or drink.
following features. • Laboratory technicians should therefore examine
➢ a more vacuolated or granular endoplasm with bacteria these preparations carefully for the presence of
and debris, but no red blood cells pathogenic parasites in addition to the
➢ a narrower, less-differentiated ectoplasm nonpathogenic E. coli.
➢ broader and blunter pseudopodia used more for feeding
than locomotion CLINICAL SYMPTOMS
➢ more sluggish, undirected movements
• Infections with E. hartmanni are typically
➢ thicker, irregular peripheral chromatin with a large,
asymptomatic.
eccentric karyosome in the nucleus
• In unstained preparations, the karyosome and surrounding TREATMENT
peripheral chromatin appear as refractile structures. The
nuclear structures are enhanced when the trophozoites are • E. coli is considered a nonpathogen, therefore,
stained. treatment is usually not indicated.
• Nuclear variations similar to those of E. histolytica and E.
hartmanni may also occur in the trophozoites of E. coli. EPIDEMIOLOGY
• In contrast to E. histolytica, red blood cell inclusions are not Who?
present in the trophozoites of E. coli. → people who have traveled to tropical places that
have poor sanitary conditions
Cyst → immigrants from tropical countries that have poor
Size Range 8-35 µm sanitary conditions
→ people who live in institutions that have poor
Shape round to spherical sanitary conditions
→ men who have sex with men
Number of Nuclei one to eight Where?
→ worldwide
Karyosome large, irregular shape, eccentric
→ areas with poor sanitation and hygiene practices
Peripheral How?
unevenly distributed
Chromatin → ingestion of infected cysts present in
contaminated food or drink
Cytoplasm coarse and granulated
PREVENTION & CONTROL
Cytoplasmic diffuse glycogen mass present in young
Inclusions cysts; may displace nuclei (often seen Individual Level
in cysts with two nuclei) to opposite ✓ adequate disposal of human feces
ends of the cyst ✓ exercising proper personal hygiene
thin chromatoid bars with pointed to ✓ protection of food from flies and cockroaches
splintered ends in young cysts
Community Level
• A thick cell wall surrounds the round to spherical cyst. ✓ proper public sanitation practices
• Occasionally, large cysts containing 16 or more nuclei may
NOTES OF INTEREST
be present.
• Iodine staining reveals dark-staining, perinuclear masses, • The morphologic differentiation of E. coli from E.
which are actually glycogen. Its location, surrounding the histolytica, as well as the pathogenicity of each of
nucleus, is more characteristic of E. coli compared to E. these parasites, was not established until the early
histolytica. 1900s.

55
 ENTAMOEBA POLECKI
MUST KNOW
→ considered as a nonpathogen
→ a parasite found in the intestines of pigs and monkeys
→ Diagnostic Stage: cyst, trophozoite
→ Infective Stage: cyst

MORPHOLOGY
Trophozoite
Size Range 8-25 µm

Motility Normal Stool: sluggish, nonprogressive

Diarrheal Stool: progressive, unidirectional

Number of
one
Nuclei

Karyosome small and central

Peripheral
fine and evenly distributed
Chromatin

Cytoplasm granular and vacuolated

Cytoplasmic ingested bacteria

Inclusions other food particles

• The single nucleus may be slightly visible in unstained

preparations. The nuclear structures have features
resembling both E. histolytica and E. coli.
• The granular and vacuolated cytoplasm resembles that of E.
coli, often containing ingested yeast, bacteria, or other food
particles.

Cyst
Size Range 10-20 µm TREATMENT
Shape spherical or oval • A combination of metronidazole (Flagyl) and
diloxanide furoate (Furamide) has successfully
Number of Nuclei one treated patients with E. polecki.
Karyosome small and central • Metronidazole alone has also been effective.

Peripheral Chromatin fine and evenly distributed EPIDEMIOLOGY

Cytoplasm granular Who?
→ relatively rare for human infections
Cytoplasmic chromatoid bars, angular or pointed
Where?
Inclusions ends in young cysts
→ only in select areas of the world, with the highest
glycogen mass in young cysts
prevalence occurring in Papua, New Guinea
Inclusion mass
How?
• A nondescript oval or round inclusion mass is seen in → ingestion of infected cysts
approximately 50% of the cysts studied. This non–glycogen- → human to human as well as pig to human
containing structure appears elusive and does not have
defined borders. The makeup of the inclusion mass is not • E. polecki has for a number of years been primarily
known. considered a parasite of pigs and monkeys.
• E. polecki can be distinguished from E. histolytica in that the • Infections with E. polecki have also been reported in
former’s cyst is consistently uninucleated, and chromatoidal Southeast Asia, France, and the United States.
bars are frequently angular or pointed. • It is interesting to note that all the reported cases of
• In stained fecal smears, the nuclear membrane and E. polecki documented in the United States in 1985
karyosome are very prominent. occurred in Southeast Asian refugees who settled in
Rochester, Minnesota.
LABORATORY DIAGNOSIS
PREVENTION & CONTROL
Specimen of Choice stool
Individual Level
CLINICAL SYMPTOMS ✓ exercising proper personal hygiene
Community Level
• Most patients with E. polecki are asymptomatic. ✓ proper public sanitation practices
• The only documented discomfort associated with ✓ education programs regarding the routes of
symptomatic patients is diarrhea. transmitting E. polecki infection

56
 ENDOLIMAX NANA
MUST KNOW
→ considered as a nonpathogen
→ occurs with the same frequency as Entamoeba coli
→ Diagnostic Stage: cyst, trophozoite
→ Infective Stage: cyst

MORPHOLOGY
Trophozoite
Size Range 5-12 µm

Motility sluggish, nonprogressive, blunt

pseudopods

Number of Nuclei one

Karyosome large, irregular, blotlike

Peripheral Chromatin absent

Cytoplasm granular and vacuolated

Cytoplasmic Inclusions bacteria

• The single nucleus may be slightly visible in unstained

preparations.
• The absence of peripheral chromatin is a key feature that
aids in the identification of E. nana trophozoites.
• They have blunt, hyaline pseudopodia, and the nucleus has
a large, irregular karyosome. Food vacuoles found in the
cytoplasm may contain bacteria
CLINICAL SYMPTOMS
Cyst
• E. nana infections are usually asymptomatic.
Size Range 4-12 µm
TREATMENT
Shape spherical, ovoid, ellipsoid
• E. nana is considered a nonpathogen, therefore,
Number of Nuclei one to four; four most common
treatment is generally not indicated.
Karyosome large, blotlike, usually central
EPIDEMIOLOGY
Peripheral
absent Who?
Chromatin
→ people living in areas of poor sanitary conditions
Cytoplasm granular and vacuolated Where?
→ in warm, moist regions of the world
Cytoplasmic chromatin granules
→ areas in which poor hygiene and substandard
Inclusions nondescript small mass
sanitary conditions exist
diffuse glycogen mass in young cysts
How?
• Although there may be one to four nuclei in an E. nana cyst, → ingestion of infected cysts in contaminated food
the most commonly seen form is the mature cyst that or drink
contains four nuclei. → fecal-oral
• The structures of these cyst nuclei are basically identical to
those seen in the trophozoite.
PREVENTION & CONTROL
• Chromatoid bars, such as those often seen in the
Entamoeba spp. cysts, are not present. Individual Level
• Cysts measure about the same size as trophozoites, and are ✓ protection of food and drink from flies and
quadrinucleated when mature. cockroaches
✓ exercising proper personal hygiene
LABORATORY DIAGNOSIS Community Level
✓ proper public sanitation practices
Specimen of Choice stool

57
 IODAMOEBA BÜTSCHLII
MUST KNOW
→ considered as a nonpathogen
→ Diagnostic Stage: cyst, trophozoite
→ Infective Stage: cyst

MORPHOLOGY
Trophozoite
Size Range 8-22 µm

Motility sluggish, usually progressive

Number of Nuclei one

Karyosome large, usually central refractive

achromatic granules may or
may not be present

Peripheral Chromatin absent

Cytoplasm coarsely granular and vacuolated

Cytoplasmic Inclusions bacteria

yeast cells
other debris

• The single nucleus consists of a large, usually central

karyosome surrounded by refractive achromatic granules,
which are often not distinct, even when the trophozoite is
permanently stained. CLINICAL SYMPTOMS
• The coarsely granular and vacuolated cytoplasm may
contain bacteria, yeast cells, or other debris. • I. bütschlii is a nonpathogenic intestinal ameba that
usually does not produce clinical symptoms.
• It is identified by its characteristic large, vesicular nucleus
with a large, central karyosome, surrounded by achromatic TREATMENT
granules.
• I. bütschlii is considered a nonpathogen, therefore,
Cyst treatment is generally not indicated.
Size Range 5-22 µm EPIDEMIOLOGY
Shape ovoid, ellipsoid, triangular, other shapes
Who?
Number of Nuclei one → people living in areas of poor sanitary conditions
Where?
Karyosome large, eccentric achromatic granules on → worldwide; but higher in tropical regions than in
one side may be present temperate regions
→ areas in which poor hygiene and substandard
Peripheral sanitary conditions exist
absent
Chromatin
How?
Cytoplasm coarsely granular and vacuolated → ingestion of infected cysts in contaminated food
or drink
Cytoplasmic well-defined glycogen mass → hand-to-mouth
Inclusions granules may be present
PREVENTION & CONTROL
• A well-defined glycogen mass with definite borders is
characteristic and is considered as an important diagnostic Individual Level
feature of the I. bütschlii cyst. ✓ exercising proper personal hygiene
• The cyst is uninucleated, and has a large glycogen body Community Level
which stains dark brown with iodine. ✓ proper public sanitation practices

LABORATORY DIAGNOSIS NOTES OF INTEREST

Specimen of Choice stool
• Iodine wet preps often prove to be of benefit, particularly in
the identification of I. bütschlii cysts.
• The glycogen mass typically picks up the iodine stain and is
thus characteristically recognizable.
• It is important to point out that the glycogen mass remains
unstained following trichrome staining, another feature that
aids in the identification of I. bütschlii cysts.
• The term Iodamoeba was coined to describe an
ameba that stains well with iodine.
• Unlike the other intestinal ameba, the nucleus of I.
bütschlii does not undergo typical division.
• The nucleus of the I. bütschlii cyst is often described
as resembling a basket of flowers in shape.
• Contaminated hog feces have been implicated as
the source of some infections with I. bütschlii.

58
 LIFE CYCLE OF NON-PATHOGENIC PROTOZOA
ENTAMOEBA COLI | ENTAMOEBA HARTMANNI | ENTAMOEBA COLI | ENDOLIMAX NANA | IODAMOEBA BÜTSCHLII

Cycle
Non-invasive Colonization
1. Entamoeba coli, E. hartmanni, E. polecki, Endolimax nana, and Iodamoeba buetschlii are generally considered
nonpathogenic and reside in the large intestine of the human host.
Diagnostic Stage
2. Both cysts and trophozoites of these species are passed in stool and considered diagnostic. Cysts are typically found in
formed stool, whereas trophozoites are typically found in diarrheal stool. Colonization of the nonpathogenic amebae
occurs after ingestion of mature cysts in fecally-contaminated food, water, or fomites.
Excystation
3. Excystation occurs in the small intestine and trophozoites are released, which migrate to the large intestine.
Replication
4. The trophozoites multiply by binary fission and produce cysts, and both stages are passed in the feces.
Infective Stage
5. Because of the protection conferred by their cell walls, the cysts can survive days to weeks in the external environment
and are responsible for transmission. Trophozoites passed in the stool are rapidly destroyed once outside the body, and
if ingested would not survive exposure to the gastric environment.

59
 ENTAMOEBA GINGIVALIS
MUST KNOW
→ considered as a nonpathogen
→ can be found in the mouth
→ Diagnostic Stage: trophozoite
→ Infective Stage: trophozoite

MORPHOLOGY
Trophozoite
Size Range 8-20 µm

Motility active, varying pseudopod appearance

Number of Nuclei one

Karyosome centrally located

Peripheral
fine and evenly distributed
Chromatin LABORATORY DIAGNOSIS
Cytoplasm finely granular Specimen of mouth scrapings, particularly
Choice from the gingival area
Cytoplasmic leukocytes
Inclusions epithelial cells • Material from the tonsillar crypts and pulmonary
bacteria abscess, as well as sputum, may also be examined.
• Vaginal and cervical material may be examined to
• The trophozoite of Entamoeba gingivalis morphologically
diagnose E. gingivalis in the vaginal and cervical
resembles that of E. histolytica.
areas.
• The pseudopods may appear long when seen at one point in
time and short and blunt the next time that they are seen. EPIDEMIOLOGY
• It is important to note that E. gingivalis is the only ameba that
ingests white blood cells. Food vacuoles that contain cellular Who?
debris (mostly leukocytes, which is characteristic of this → in all populations that have been studied for its
species) and bacteria are numerous. presence
Where?
Cyst → areas in which poor hygiene conditions exist
• There is no known cyst stage of E. gingivalis. How?
→ mouth-to-mouth (kissing)
CLINICAL SYMPTOMS → droplet contamination, which may be transmitted
through contaminated drinking utensils
• Infections of E. gingivalis occurring in the mouth and in the
genital tract typically produce no symptoms. • They are abundant in cases of oral disease.
• Nonpathogenic E. gingivalis trophozoites are frequently • Transmission is most probably direct: through
recovered in patients suffering from pyorrhea alveolaris. kissing, droplet spray, or by sharing utensils.
• It appears that the trophozoites thrive under disease
conditions but do not produce symptoms of their own.
PREVENTION & CONTROL
Individual Level
TREATMENT ✓ improved oral hygiene accomplished by the proper
care of the teeth and gums
• E. gingivalis is considered a nonpathogen, therefore,
✓ prompt removal of IUDs in infected patients
treatment is generally not indicated.
✓ avoidance of mouth-to-mouth kissing
NOTES OF INTEREST Community Level
✓ education programs for E. gingivalis transmission
• Discovered in 1849, E. gingivalis was the first ameba
recovered from a human specimen.

60
 ENTAMOEBA GINGIVALIS
LIFE CYCLE

Notes
• Mode of Transmission: mouth-to-mouth (kissing); droplet contamination, which may be transmitted through
contaminated drinking utensils
• Diagnostic Stage: trophozoite
• Infective Stage: trophozoite
• E. gingivalis, as the name implies, typically lives around the gum line of the teeth in the tartar and gingival pockets of
unhealthy mouths.
• In addition, E. gingivalis trophozoites have been known to inhabit tonsillar crypts and bronchial mucus.
• It is particularly important to diagnose E. gingivalis and E. histolytica correctly because both organisms may be found in
the sputum and in pulmonary abscesses.
• E. gingivalis may also be found in the mouths of individuals who practice good oral hygiene.
• Existing as a scavenger, the E. gingivalis trophozoites feed on disintegrated cells and multiply by binary fission.
• Characteristically delicate, these trophozoites will not survive following contact with stomach juices.
• E. gingivalis trophozoites have also been recovered in vaginal and cervical specimens from women who are using
intrauterine devices (IUDs). Spontaneous disappearance of the trophozoites seems to occur following removal of the
IUD.

61
 NAEGLERIA FOWLERI
MORPHOLOGY
Notes Ameboflagellate (Flagellate Form)

• Naegleria spp. are free-living protozoans with two vegetative

forms: an ameba (trophozoite form), and a flagellate
(swimming form).
• A dormant cyst form is produced when conditions are not
favorable. Transformation from the trophozoite to the
flagellate form may facilitate more rapid movement toward
food sources.
• There are two forms of trophozoites of Naegleria fowleri:
ameboid and ameboflagellate, only the former of which is
found in humans.

Size Range 7-15 µm

Shape pear-shaped

Motility jerky, spinning

Number of Nuclei one

Karyosome large, central

Peripheral Chromatin absent

Cytoplasm granular, usually

Ameboid Trophozoite vacuolated

• Two whiplike structures that assist select parasites

in locomotion known as flagella extend from the
broad end of the organism.

Cyst

Size Range 8-22 µm

Motility sluglike, blunt pseudopods

Number of Nuclei one

Size Range 9-12 µm
Karyosome large and usually centrally Shape round
located
Motility jerky, spinning
Peripheral Chromatin absent
Number of Nuclei one
Cytoplasm granular, usually vacuolated

• The typical ameboid trophozoite of N. fowleri appears Karyosome large, central

elongate.
Peripheral Chromatin absent
• The ameboid trophozoites measure 10 to 35 µm but when
rounded are usually 10 to 15 µm in diameter. In culture, Cytoplasm granular, usually
trophozoites may get over 40 µm. vacuolated

62
 NAEGLERIA FOWLERI
LIFE CYCLE
Notes
• Mode of Transmission: ingestion of
fecally-contaminated material; venereal
transmission through fecal-oral contact
or direct colonic inoculation through
contaminated enema equipment
• Diagnostic Stage: trophozoite,
flagellate
• Infective Stage: trophozoites
• Naegleria spp. are thermophilic
organisms which thrive best in hot
springs and other warm aquatic
environments.
• Both nonpathogenic and pathogenic
forms exist.
➢ Only Naegleria fowleri has been
reported to consistently cause
disease in humans, although some
non-fowleri species may cause
opportunistic infections.
• Naegleria fowleri has three stages,
cysts, trophozoites, and flagellated
forms, in its life cycle.
• The trophozoites replicate by
promitosis (nuclear membrane
remains intact), or by simple binary
fission, and can turn into temporary
non-feeding flagellated forms, which
usually revert back to the trophozoite
stage.
• Trophozoites infect humans or
animals by penetrating the nasal
mucosa and migrating to the brain
via the olfactory nerves.
• N. fowleri trophozoites are found in
cerebrospinal fluid (CSF) and tissue,
while flagellated forms are
occasionally found in CSF. Cysts are
not seen in brain tissue.
Cycle
• The ameboid trophozoites transform
Three Forms into flagellate trophozoites in vitro
after being transferred to water from
1. Cysts a tissue or culture.
2. Trophozoite Forms • The flagellate trophozoites do not
3. Flagellate Forms divide but rather lose their flagella
Promitosis and convert back into the ameboid
form, in which reproduction resumes.
4. The trophozoites replicate by promitosis (nuclear membrane remains
intact). • The cyst form is known to exist only in
the external environment.
Infective Stage
• It appears that the entire life cycle of
5. Naegleria fowleri is found in fresh water, soil, thermal discharges of power N. fowleri, which consists of the
plants, geothermal wells, and poorly chlorinated recreational and tap amebic trophozoites converting to
water. cysts and flagellates and then back to
• Trophozoites can turn into temporary non-feeding flagellated forms amebic trophozoites, occurs in the
which usually revert back to the trophozoite stage. external environment.
• Trophozoites infect humans or animals by penetrating the nasal
mucosa, usually during swimming or sinus irrigation. • Humans primarily contract this
ameba by swimming in
Brain Migration contaminated water.
6. Naegleria fowleri migrates to the brain via the olfactory nerves causing • The ameboid trophozoites enter the
PAM. human body through the nasal
Diagnostic Stage mucosa and often migrate to the
7. Naegleria fowleri trophozoites are found in cerebrospinal fluid (CSF) and brain, causing rapid tissue
tissue, while flagellated forms are occasionally found in CSF. Cysts are not destruction.
seen in brain tissue. • Some infections may be caused by
inhaling dust infected with N. fowleri.

63
 NAEGLERIA FOWLERI
PATHOGENESIS & CLINICAL SYMPTOMS
• Asymptomatic
▪ Patients who contract N. fowleri resulting in colonization of the
nasal passages are usually asymptomatic.
• Primary Amebic Meningoencephalitis (PAM)
→ occurs when the ameboid trophozoites of N. fowleri invade the
brain, causing rapid tissue destruction
▪ Patients may initially complain of fever, headache, sore
throat, nausea, and vomiting.
▪ Symptoms of meningitis rapidly follow, including stiff neck
and seizures.
▪ In addition, the patient will often experience smell and
taste alterations, blocked nose, and Kernig’s sign.
➢ Kernig’s Sign
→ defined as a diagnostic sign for meningitis, where
the patient is unable to fully straighten his or her
leg when the hip is flexed at 90 degrees because of
hamstring stiffness
→ In untreated patients, death usually occurs 3 to 6 days after
onset.
→ Postmortem brain tissue samples of these patients reveal the
typical ameboid trophozoites of N. fowleri.
• N. fowleri is the causative agent of a rare but rapidly destructive
and fatal meningoencephalitis termed primary amebic
meningoencephalitis (PAM).
• In contrast to granulomatous amebic encephalitis (GAE) which is
predominantly an opportunistic infection, PAM usually occurs in
previously healthy adults with a history of swimming.
• N. fowleri is able to survive in elevated temperatures and
reproduces rapidly in temperatures above 30°C. Aside from
naturally occurring hot springs, warm geothermal plant effluent
into lakes and streams can lead to proliferation of amebae.
• Most cases of PAM have occurred in young, healthy persons who
swim in contaminated water.
• The route of entry is through invasion of organisms through the
olfactory bulb after accidental inhalation of water containing the
organisms.
▪ The sustentacular cells of the olfactory neuroepithelium are
thought to phagocytose the amebae and transport these
through the cribriform plate and into the brain.
▪ Multiple mechanisms then come into play, producing a
cytopathic effect on host tissues.
▪ These mechanisms include secretion of lytic enzymes,
membrane pore-forming proteins, factors which induce
apoptosis, and direct feeding on cells by the amebae.
• In humans, PAM presents as fever, nausea, vomiting, headache,
nuchal rigidity, and mental status changes, with rapid
progression to coma and death.
• Characteristic cerebrospinal fluid findings include elevated white
blood cell count with neutrophilic predominance, high protein,
and low glucose.
• Post-mortem examination of infected brain shows hemorrhagic
necrosis, particularly of the olfactory bulbs, congestion and
edema of neural tissue. Leptomeninges are inflamed and
congested as well. Microscopic examination shows a
fibrinopurulent exudate consisting mostly of neutrophils in the
leptomeninges and brain tissue, and pockets of amebae with
scant inflammatory exudates in necrotic areas.
• Death usually occurs as a result of cerebral or cerebellar
herniation as a result of increased intracranial pressure.
64
LABORATORY DIAGNOSIS
Specimen of Choice cerebrospinal fluid
• Preparing and scanning saline and iodine wet
preparations of the CSF are recommended.
• Samples of tissues and nasal discharge may
also be examined.
• Clinical specimens may be cultured.
• Naegleria fowleri ameboid trophozoites show a
characteristic trailing effect when placed on
agar plates that have been previously
inoculated with gram-negative bacilli.
• Diagnosis of PAM is usually suspected in
persons with a compatible history of exposure
and a rapidly progressive meningoencephalitis.
• Aspirates from suspected infections, when
introduced into bacteria-seeded agar culture
medium, will exhibit active trophozoites within
24 hours.
• Flagellation tests have poor sensitivity for
identification since amebae which test
negative have been subsequently identified as
Naegleria spp. and Naegleria fowleri with more
sensitive and specific molecular techniques
such as PCR and immunostaining.
• Serology utilizing ELISA is less useful in
diagnosing active infection since healthy
individuals especially in endemic areas have
been shown to have positive antibody titers.
TREATMENT
• Most persons infected with Naegleria die prior to
institution of effective treatment.
• Symptoms of PAM are indistinguishable from
bacterial meningitis.
• Initial CSF results are suggestive of a bacterial
etiology, and so patients are typically treated with
antibiotics which have no activity against
Naegleria.
• Unfortunately, medications used to treat
meningitis and amebic infections are ineffective
against N. fowleri.
• There is evidence, however, that prompt and
aggressive treatment with amphotericin B may be
of benefit to patients suffering from infections with
N. fowleri, despite its known toxicity.
• In rare cases, amphotericin B in combination with
rifampin or miconazole has also proved to be an
effective treatment. Amphotericin B and
miconazole damage the cell wall of Naegleria,
inhibiting the biosynthesis of ergosterol and
resulting in increased membrane permeability,
which causes nutrients to leak out of the cells.
• Rifampicin inhibits RNA synthesis in the amoeba by
binding to beta subunits of DNA-dependent RNA
polymerase, which in turn blocks RNA transcription.
• A person can survive if signs are recognized early
but, if not, PAM almost always results in death.
 NAEGLERIA FOWLERI
EPIDEMIOLOGY PREVENTION & CONTROL
Who? Individual Level
→ young males ✓ avoiding immersion of the head and
→ people participating in activities such as diving into the water accidental inhalation of water in endemic
and playing in the sediment at the bottom of lakes and rivers areas and in hot springs
Where? ✓ repairing of cracks found in the walls of pools,
→ in warm bodies of water, including lakes, streams, ponds, and hot tubs, and baths
swimming pools Community Level
How? ✓ posting off-limits signs around known sources
→ trophozoite through the nasal mucosa of contamination
→ inhalation of contaminated dust ✓ educating the medical community and
→ sniffing contaminated water may transmit N. fowleri public
✓ appropriate decontamination of swimming
Notes water

• Prevalence is higher in the summer months of the year. NOTES OF INTEREST

• In addition to water sources, there have been cases of
contaminated dust. One such case occurred in Nigeria, a country
that has a warm climate.
• Distribution of Naegleria in freshwater lakes and ponds has been
correlated with physical, chemical, and biological parameters.
• Strains have been frequently isolated from thermal effluents, hot
springs, and water with naturally or artificially elevated
temperatures.
• Fecal coliform contamination provides a ready food source for
ameba, and may increase the risk of infection due to higher
density of organisms.
• Studies on local Naegleria have identified a new species which is
morphologically indistinguishable but biochemically distinct from
other known species.
• Isolates from a thermally-polluted stream, an artificially-heated
swimming pool, and from the brain aspirate of a young patient
have all yielded a single species, N. philippinensis. This has been
extensively studied by Castro et al., and Matias et al.
• Only one case of PAM has been reported locally, in a young male
with a history of swimming in fresh water. He responded well to
amphotericin B infusion.
• Two Philippine isolates of Naegleria (NSzu and RITM strains) have
been evaluated for pathogenicity. Massive doses of amebae
successfully established infection in the brain and caused death
in some mice within two to six days post-inoculation.
• Clinical features of infection and histopathology were compatible
with PAM.
• The first case of PAM was reported by Carter
and Fowleri, for whom the ameba is named, in
1965 in Australia, and by Butt and Patras in 1966
in the United States.
• One noteworthy species of Naegleria that
could possibly infect humans in the future is
known as N. australiensis.
▪ This organism exists in the environment in
Asia, Australia, Europe, and United States.
▪ N. australiensis has been found to be
pathogenic in mice that have been
exposed to this parasite by intranasal
instillation.
• A number of methods have been studied in
recent years aimed at classifying, identifying
and speciating Naegleria.
▪ These laboratory techniques include PCR
assay, monoclonal antibody testing, flow
cytometry, and DNA hybridization.
▪ In addition, a method designed to aid in
taxonomic classification, called DNA
restriction fragment length polymorphism
(RFLP), has been studied.
▪ Results of all of these tests to date have
been favorable.
▪ Further studies, however, have been
recommended

65
 ACANTHAMOEBA SPECIES
Notes

• Acanthamoeba is a ubiquitous, free-living ameba that is the etiologic agent of Acanthamoeba keratitis (AK) and
granulomatous amebic encephalitis (GAE).
• Acanthamoeba is characterized by an active trophozoite stage with characteristic prominent “thorn-like” appendages
(acanthopodia); and a highly resilient cyst stage into which it transforms when environmental conditions are not
favorable.
• It is an aquatic organism that is found in a myriad of natural and artificial environments and can survive even in contact
lens cleaning solutions.
• Motile trophozoites feed on gram-negative bacteria, blue-green algae, or yeasts and reproduce by binary fission, but
can also adapt to feed on corneal epithelial cells and neurologic tissue through phagocytosis and secretion of lytic
enzymes.

MORPHOLOGY
Trophozoite Cyst

Size Range 12-45 µm Size Range 8-25 µm

Motility sluggish, spinelike pseudopods Shape roundish with ragged edge

Number of Nuclei one Number of Nuclei one

Karyosome large Karyosome large, central

Peripheral Chromatin absent Peripheral Chromatin absent

Cytoplasm granular and vacuolated Cytoplasm disorganized, granular,
• Spinelike pseudopods, known as acanthopodia, project sometimes vacuolated
outward from the base of the organism.
Other Features
• Morphologically, Acanthamoeba trophozoites exhibit a double cell walll—smooth
characteristic single large nucleus with a centrally-located, inner cell wall and outer
densely staining nucleolus; a large endosome; finely jagged cell wall
granulated cytoplasm; and a large contractile vacuole.

66
 ACANTHAMOEBA SPECIES
PATHOGENESIS & CLINICAL SYMPTOMS
GRANULOMATOUS AMEBIC ENCEPHALITIS (GAE)
• CNS infections with Acanthamoeba are also known
granulomatous amebic encephalitis (GAE).
• Symptoms of this condition develop slowly over time and include
headaches, seizures, stiff neck, nausea, and vomiting.
• Granulomatous lesions of the brain are characteristic and may
contain both Acanthamoeba trophozoites and cysts.
• On occasion, Acanthamoeba spp. invade other areas of the body,
including the kidneys, pancreas, prostate, and uterus, and form
similar granulomatous lesions.
AMEBIC KERATITIS
• Acanthamoeba infections of the cornea of the eye are known as
amebic keratitis.
• Common symptoms include severe ocular pain and vision
problems.
• The infected tissue of the cornea may contain Acanthamoeba
trophozoites and cysts.
• Perforation of the cornea may result, as well as subsequent loss of
vision.
• Acanthamoeba was first described as an opportunistic ocular
surface pathogen causing keratitis in 1974.
• AK is associated with the use of improperly disinfected soft contact
lenses, particularly those which are rinsed with tap water or
contaminated lens solution.
• An immunocompromised state contributes to increased
susceptibility to infection and may lead to disseminated disease in
the lungs and brain (GAE).
• Symptoms of AK include severe ocular pain and blurring of vision.
Corneal ulceration with progressive corneal infiltration may occur.
• Primary amebic infection or secondary bacterial infection may lead
to hypopyon formation. Progression of infection may cause scleritis
and iritis and may ultimately lead to vision loss. Major differentials
which need to be ruled out include fungal and herpetic keratitis.
• Acanthamoeba was documented as the causative agent of human
GAE by Stamm in 1972.
• Amebae were demonstrated in brain sections of a GAE patient using
indirect fluorescence microscopy.
• GAE usually occurs in immunocompromised hosts including the
chronically ill and debilitated, and those on immunosuppressive
agents such as chemotherapy and anti-rejection medications.
• Signs and symptoms of GAE are generally related to destruction of
brain tissue and the associated meningeal irritation.
• Systemic manifestations early in the course include fever, malaise,
and anorexia. Neurologic symptoms may include increased
sleeping time, severe headache, mental status changes, epilepsy,
and coma.
• Neurologic findings depending on the location of the lesions include
hemiparesis, blurring of vision, diplopia, cranial nerve deficits, ataxia,
and increased intracranial pressure.
• Entry of Acanthamoeba into the central nervous system is still
incompletely understood. From a primary site of infection in the skin
or lungs, the likely route of invasion is hematogenous. Direct
infection through the olfactory valves has also been proposed, but
not conclusively demonstrated.
• The incubation period from initial inoculation is approximately 10
days, with a subacute and chronic clinical course of infection that
lasts for several weeks to several months.
• The clinical manifestations of disease include decreased sensorium,
altered mental status, meningitis, and neurologic deficits. The
natural course of the disease eventually results in coma and death.
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LABORATORY DIAGNOSIS
Specimen of Choice cerebrospinal fluid
as
• Brain tissue may also be examined.
• Corneal scrapings are the specimen of
choice for recovery of Acanthamoeba
infections of the eye.
➢ Suspected corneal scrapings may be
cultured on non-nutrient agar plates
seeded with gram-negative bacteria
(specifically, a viable strain of E. coli). The
bacteria serve as a source of food for the
parasites. As the Acanthamoeba
organisms feed, they produce a set of
marks (known as tracks) on the agar.
➢ Histologic examination of corneal
scrapings may also recover
Acanthamoeba. Although primarily used
to detect fungi in clinical specimens,
calcofluor white may be used to stain
Acanthamoeba cysts present in corneal
scrapings.
• Indirect immunofluorescent antibody
staining is the technique of choice for
speciating Acanthamoeba.
ACANTHAMOEBA KERATITIS
• Acanthamoeba keratitis is diagnosed by
epithelial biopsy or corneal scrapings for
recoverable ameba with characteristic
staining patterns on histologic analysis.
• Amebae have also been isolated from the
contact lens and lens solution of patients.
• Species-specific identification can be made
from culture and molecular analysis through
PCR. Known species that have caused AK
include A. castellani, A. culbertsoni, A.
hutchetti, A. polyphaga, and A. rhysoides.
GRANULOMATOUS AMEBIC ENCEPHALITIS (GAE)
• Diagnosis of GAE is usually made post-
mortem in most cases.
• The rarity of the disease and unfamiliarity of
most physicians with the pathogen
contribute to frequently missed diagnosis.
• Signs and symptoms of disease are usually
attributed to more common differentials.
• Moreover, recovery of ameba from
cerebrospinal fluid is exceedingly rare, and
imaging results are generally nonspecific.
• Immunocompromised patients such as
those with AIDS are at the highest risk for
acquiring GAE.
• While opportunistic infections of the central
nervous system such as Cryptococcus
meningitis and toxoplasmosis are much
more common than GAE, the lack of
response despite appropriate treatment
should prompt a more thorough evaluation
for more esoteric organisms.
• Specific diagnosis depends on
demonstrating the trophozoites or cysts in
tissues using histopathologic stains and
microscopy.
• The organisms can rarely be demonstrated
in the cerebrospinal fluid and can be
cultured for further studies.
 ACANTHAMOEBA SPECIES
LIFE CYCLE

Cycle
Two Stages
1. Cysts
2. Trophozoites
Mitosis
3. The trophozoites replicate by
mitosis (nuclear membrane does
not remain intact).
Infective Stage
4. The trophozoites are the infective
forms, although both cysts and
trophozoites gain entry into the
body through various means.
5. through the eye
6. the nasal passages to the lower
respiratory tract
7. ulcerated or broken skin
Pathogenic Stage
8. When Acanthamoeba spp. enters
the eye, it can cause severe keratitis
in otherwise healthy individuals,
particularly contact lens users.
When it enters the respiratory
system or through the skin, it can
invade the central nervous system
by hematogenous dissemination
causing the following in individuals
with compromised immune
systems.
9. granulomatous amebic
encephalitis (GAE)
10. disseminated disease
11. skin lesions
Diagnostic Stage
12. Acanthamoeba spp. cysts and
trophozoites are found in tissue.
Notes
• Diagnostic Stage: cyst, trophozoite
• Infective Stage: trophozoite
• The trophozoites and cysts of Acanthamoeba convert between these two
morphologic forms in the external environment.
• Humans may acquire Acanthamoeba in one of two ways.
➢ One route consists of aspiration or nasal inhalation of the organisms.
Trophozoites and cysts enter via the lower respiratory tract or through
ulcers in the mucosa or skin. These organisms often migrate via
hematogenous spread—that is, transported through the bloodstream—and
invade the central nervous system (CNS), causing serious CNS infections.
➢ The second route of infection consists of direct invasion of the parasite in
the eye. Two groups of individuals are at risk for direct eye invasion, contact
lens wearers and those who have experienced trauma to the cornea.
• Contact lens wearers who use homemade, nonsterile saline solutions that are
contaminated with Acanthamoeba typically suffer a serious eye infection,
known as Acanthamoeba keratitis.
• It is important to note that unlike N. fowleri, which is associated with swimming
or bathing in contaminated water, Acanthamoeba spp. infection is not
associated with water but rather with contaminated saline.
• There are currently 10 species of Acanthamoeba known to infect humans.
Acanthamoeba castellanii has been identified as the species responsible for
most CNS and eye infections in humans.
• No flagellated stage exists as part of the life cycle.
• The trophozoites replicate by mitosis (nuclear membrane does not remain
intact).
• The trophozoites are the infective stage, although both cysts and trophozoites
gain entry into the body through various means. Entry can occur through the
eye, the nasal passages to the lower respiratory tract, or ulcerated or broken
skin.
• The presence of naturally-occurring bacterial endosymbionts in
Acanthamoeba spp. has been reported. Although the presence of bacterial
symbionts is widespread among small, free-living amebae, the significance of
this association is not known.
• Recently, Acanthamoeba spp. have been implicated as possible reservoir
hosts for medically important bacteria such as Legionella spp., mycobacteria,
and gram-negative bacilli such as E. coli.

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 ACANTHAMOEBA SPECIES
EPIDEMIOLOGY
Who?
→ patients who are immunocompromised or debilitated
→ people with poor hygiene practices, especially the use of
homemade saline rinsing solutions
→ people using contact lens wearers, particularly those
wearing soft contacts
Where?
→ worldwide
How?
→ to the eyes through contact lens use, cuts, or skin wounds
→ by being inhaled into the lungs
Notes
• Acanthamoeba spp. have a protean distribution, having
been isolated from a multitude of natural and artificial
aquatic environments including fresh and salt water,
sewage, hospital equipment, and contact lenses and lens
solution.
• De Jonckheere first diagnosed Acanthamoeba GAE in a
living patient in 1991. Previously, diagnosis of GAE was post-
mortem.
• AK was recognized earlier in the 1970s and has been
reported in the United States, Europe, South America, and
Asia.
• The first case of AK was recognized in the Philippines in the
1990s from a patient from the Philippine General Hospital,
and samples obtained from the patient was shown to cause
GAE in mice.
• Multiple environmental isolates have likewise been well-
characterized from all over the Philippines, including a few
containing endosymbionts.
• Animals, including rabbits, beavers, cattle, water buffalo,
dogs, and turkeys, have been known to contract
Acanthamoeba infections.
• Just as in humans, immunocompromised animals appear to
contract fatal CNS infections.
PREVENTION & CONTROL
Individual Level
✓ avoiding immersion of the head and accidental inhalation
of water in endemic areas and in hot springs
✓ repairing of cracks found in the walls of pools, hot tubs, and
baths
Community Level
✓ posting off-limits signs around known sources of
contamination
✓ educating the medical community and public
✓ appropriate decontamination of swimming water
69
TREATMENT
• Because of the slow progression of GAE, most patients
who suffer from it die, not only before an accurate
diagnosis may be made, but also before experimental
treatments can be administered and studied.
• There is some evidence to suggest that sulfamethazine
might be a suitable treatment.
• Cases of Acanthamoeba keratitis have successfully
been treated with several medications that include
itraconazole, ketoconazole, miconazole, propamidine
isethianate, and rifampin.
• Of all these agents, propamidine appears to have the
best documented success record.
• The key to successful treatment to eye infections is to
begin treatment immediately once the infection has
been diagnosed.
• Medical treatment of AK has been met with increasing
success in recent years.
• While historically, only surgical excision of the infected
cornea with subsequent corneal transplantation was
curative, early recognition of AK coupled with
aggressive combination anti-amebic agents can
preclude the need for extensive surgery.
• D’Aversa and his colleagues have achieved acceptable
results with clotrimazole combined with pentamidine,
isethionate, and neosporin.
• Other agents that have been used include
polyhexamethylene biguanide, propamidine,
dibromopropamidine isethionate, neomycin,
paromomycin, polymyxin B, ketoconazole, miconazole,
and itraconazole.
• Topical corticosteroids should be avoided, as this
retards the immune response.
• Advanced AK usually requires debridement, but
complete excision of the cornea can be avoided if the
infection is confined to more superficial areas.
• Deep lamellar keratectomy is the procedure of choice.
• Clinically apparent neurologic disease in GAE usually
heralds a fatal outcome within 3 to 40 days.
• A few patients have shown good responses to
combinations of amphotericin B, pentamidine
isethionate, sulfadiazine, flucytosine, fluconazole or
itraconazole.
• One liver transplant patient survived after
decompressive frontal lobectomy and treatment with
amphotericin, cotrimoxazole, and rifampin.
• Poor prognostic factors include severe
immunosuppression and advanced disease.
 ACANTHAMOEBA SPECIES
NOTES OF INTEREST
• Acanthamoeba shares many characteristics with the gram-negative bacteria Pseudomonas aeruginosa, which
frequently occurs in standing water as an eye pathogen, but they are usually not recovered simultaneously from the
same patient.
• It is believed that P. aeruginosa inhibits the activity of Acanthamoeba spp.
• Acanthamoeba has been rarely known to infect areas of the body other than those typically reported.
• An interesting case involved cutaneous lesions filled with Acanthamoeba trophozoites and cysts on the trunk, legs, and
arm of a patient suffering from AIDS.
• The patient also presented with brain lesions that did not show the Acanthamoeba organisms. Another case involved
Acanthamoeba invasion of bone following a graft procedure. In this case, the patient subsequently developed
osteomyelitis.
• Several newer testing methods aimed at differentiating the strains of Acanthamoeba have been studied, including
monoclonal antibodies and flow cytometry.
• In addition, DNA RFLP tests have been performed to aid in taxonomic classification of Acanthamoeba spp.
• Although additional testing with all these techniques has been suggested, available tests have shown promising results.

REFERENCES
➢ Belizario, et. al. (2013). Medical
Parasitology in the Philippines. The
University of The Philippines Press
➢ Zeibig. (2013). Clinical Parasitology: A
Practical Approach. Elvisier Inc.

TRANSCRIBED BY
➢ Bautista, MD

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