The Intestinal Protozoa

A. Introduction

1. The Phylum Protozoa is classified into four major subdivisions according to the
methods of locomotion and reproduction.

a. The amoebae (Superclass Sarcodina, Class Rhizopodea move by

means of pseudopodia and reproduce exclusively by asexual binary
division.
b. The flagellates (Superclass Mastigophora, Class Zoomasitgophorea)
typically move by long, whiplike flagella and reproduce by binary fission.

c. The ciliates (Subphylum Ciliophora, Class Ciliata) are propelled by rows

of cilia that beat with a synchronized wavelike motion.

d. The sporozoans (Subphylum Sporozoa) lack specialized organelles of

motility but have a unique type of life cycle, alternating between sexual
and asexual reproductive cycles (alternation of generations).

e. Number of species - there are about 45,000 protozoan species; around

8000 are parasitic, and around 25 species are important to humans.

2. Diagnosis - must learn to differentiate between the harmless and the medically
important. This is most often based upon the morphology of respective
organisms.

3. Transmission - mostly person-to-person, via fecal-oral route; fecally

contaminated food or water important (organisms remain viable for around 30
days in cool moist environment with few bacteria; other means of transmission
include sexual, insects, animals (zoonoses).

B. Structures

1. trophozoite - the motile vegetative stage; multiplies via binary fission; colonizes
host.

2. cyst - the inactive, non-motile, infective stage; survives the environment due to
the presence of a cyst wall.

3. nuclear structure - important in the identification of organisms and species

differentiation.

4. diagnostic features

a. size - helpful in identifying organisms; must have calibrated objectives on

the microscope in order to measure accurately.

b. type of motility - directional or non-directional; sluggish or fast.

c. cytoplasmic inclusions - chromatoid bars (coalesced RNA); red blood

cells; food vacuoles containing bacteria, yeast, etc.

d. appearance of cytoplasm - smooth & clean or vacuolated.

5. endosome - also called the karyosome, this is a mass of chromatin within

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 nucleus. The size, shape, and location of this structure are helpful in
identification of organisms.

6. chromatin - nuclear DNA

7. chromatoid body or bar - coalesced RNA within the cytoplasm in the cyst stage.
This is not always present, but when it is, its size and shape are helpful in
determining species identification.

II. Class Lobosea - The Amoebae

A. Life cycle -

1. The definitive host ingests the infective cyst stage from fecal contamination in
environment.

2. The cyst passes into the small intestine & excystation occurs with transformation
to the trophozoite stage.

3. Trophozoites in the large intestine colonize the host by multiplying asexually via
binary fission. They can remain in the lumen or invade the wall of the intestine
(pathogenic species only) & multiply, from here they can be transported via the
circulation to other organs (liver, lungs, etc.).

4. Cysts and trophozoites are passed in the feces of the infected host.

5. Infective stage - the mature cyst.

6. Diagnostic stage - the trophozoite or cyst in stool or tissue specimens.

B. Genus Entamoeba - contains the most important of the amoebae causing disease in
humans.

1. Entamoeba histolytica

Entamoeba histolytica trophozoites Entamoeba histolytica trophozoite

with ingested red blood cells with ingested red blood cells

Entamoeba histolytica cyst Entamoeba histolytica cyst E. histolytica cyst

with chromatoid bar with chromatoid bar (iodine stain)

a. Epidemiology - Occurs worldwide; the highest incidence and prevalence

is found in areas with poor sanitation where as many as 80% of a
population may be infected. Highest in children >5 years of age; more

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 prevalent in males than in females; common in mental hospitals, prisons,
orphanages.

b. Pathology and Clinical Manifestations - the most pathogenic of all;

causes amoebic dysentery; can become extra-intestinal (liver, lungs,
etc.; can be fatal.

c. Morphology & Laboratory Identification - the trophozoite ranges between

12 and 30 microns in diameter. Its nucleus has an even distribution of
peripheral chromatin and a small, compact, centrally located karyosome.
The cytoplasm is typically smooth and granular with inclusions, if
present, containing red blood cells. The cyst ranges between 10 and 30
microns in diameter and contains four nuclei when mature. Cigar-shaped
chromatoid bars may be present in some cysts.

d. Treatment - depends upon location of infection (lumen vs. tissue)

e. Distribution - worldwide, mostly in tropics and sub-tropics.

f. Note - chronic infections may last for years; they are often confused with
other conditions (colitis, cancer). Hepatic abscess is the most common
and dangerous complication.

2. Entamoeba hartmanni

Entamoeba hartmanni trophozoite Entamoeba hartmanni trophozoite

trichrome stain iron-hematoxylin stain

Entamoeba hartmanni cyst (iodine stain)

a. Formerly called the small race of Entamoeba histolytica.

b. Technologists must be able to differentiate this organism from E.

histolytica because E. hartmanni is non-pathogenic.

c. Morphology & Laboratory Identification - this organism is very similar to

E. histolytica. The difference lies in the respective sizes of the
organisms. Trophozoites of Entamoeba hartmanni will measure less
than 12 microns, while cysts will measure less than 10 microns. It is
strongly recommended that more than one organism be measured when
determining size.

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 2. Entamoeba coli

Entamoeba coli trophozoite E. coli trophozoite E. coli cyst E. coli cyst (iodine)

a. Morphology - trophozoites range from 10 to 35 microns in diameter;

cysts range from 10 to 30 microns in diameter and contain 8 to 16 nuclei
when mature; the nucleus exhibits an eccentric karyosome with irregular,
coarse chromatin. The cytoplasm is heavily vacuolated, containing
yeast, bacteria, and debris.

b. Significance - this is a harmless commensal but must be differentiated

from the pathogens.

2. Entamoeba gingivalis

Entamoeba gingivalis trophozoite

a. Infective site - in the mouth; the organism thrives in diseased gums, but
is not considered a causal agent or pathogen. If swallowed, it is
destroyed in stomach.

b. Transmission - contact with fomites (drinking glasses, eating utensils,

etc.; kissing.

c. Morphology - resembles E. histolytica, but has no cyst stage. It is the

only species, which ingests leucocytes.

C. Other amoebae of significance

1. Endolimax nana

Endolimax nana trophozoite E. nana cyst E. nana cyst (iodine)

a. Occurrence - occurs in about 14% of the US population; 21% worldwide.

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 b. Pathogenicity - none.

c. Morphology - trophozoites range from 5 to 10 microns in diameter. The

nucleus contains a large, blot-like karyosome and has little or no
peripheral chromatin. Cysts are usually sub-oval, measuring 4 to 6 by 6
to 10 microns

2. Iodamoeba butschlii

Iodamoeba butschlii trophozoite I. Butschlii cyst I. Butschlii cyst (iodine)

a. Pathogenicity - none.
b. Morphology often called iodine cyst - striking characteristic is large
glycogen vacuole - stains dark brown with iodine.

3. Naegleria fowleri

Naegleria fowleri trophozoite in CSF N. fowleri trophozoites

a. Classification - an ameboflagellate and a free-living organism; it

alternates between amoeboid and bi-flagellated forms; only the
amoeboid form is found in tissues. Disease occurs during summer
months. Occurs only in fresh water, can tolerate chlorinated swimming
pools.

b. Life cycle - the amoeba gains entry via the nasal mucosa, usually during
a swimming event; it moves along the olfactory nerve, gaining access to
the brain via the cribriform plate. It rapidly colonizes the brain, and cases
are invariably fatal. Infections do not spread form person-to-person.

c. Symptoms - Symptoms are dramatic and rapidly progressive. Headache,

fever, nausea & vomiting occur within 1 to 2 days. Meningoencephalitis,
irrational behavior, coma & death usually occur within 9 days of
exposure.

d. Diagnosis - Usually made at autopsy. CSF contains a large number of

segmenters with no bacteria present. Motile amoebae can be seen in
wet mounts of CSF. Early diagnosis is critical. CSF specimens can be
plated onto non-nutrient agar containing a lawn of bacteria; amoebae,
which grow out, can be transferred into sterile filtered water to determine

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 if the organisms present transform into the bi-flagellated stage.

e. Treatment - Amphotericin B (Intravenous and Intrathecal) with rifampin is

the only regimen to date that has proven successful in saving a patient.

4. Acanthamoeba spp.

Acanthamoeba spp. trophozoite

a. Life cycle - also a free-living amoeba. The amoeba reaches the brain
hematogenously after entering a wound or lesion on the skin. More
commonly, the organism is associated with getting into eyes via
contaminated or homemade cleaning solutions.

b. Symptoms - slow onset (10 or more days). Presents as chronic,

granulomatous lesions in brain. In eye lesions, the infection resembles a
herpes virus infection.

c. Acanthamoeba keratitis - affects healthy persons; sometimes associated

with eye trauma; most recently has been associated with users of
extended-wear contact lenses. Cysts are resistant to drying and
disinfection via chlorine.

III. Superclass Mastigophora - the flagellates; members of this group can inhabit mouth,
bloodstream, tissues, gastrointestinal, or urogenital tracts.

A. Members:

Giardia lamblia
Dientamoeba fragilis
Chilomastix mesnili
Trichomonas hominis
Retortamonas intestinalis*
Enteromonas hominis*

* will not be discussed- nonpathogenic, very rare, and of no medical significance.

B. Morphological Characteristics

1. Flagellum(ae) - characteristic organelle of locomotion. It is an extension of

ectoplasm and resembles a tail; moves with a whip-like motion.

2. Axostyle - a supporting mechanism; a rod-shaped structure; not all Genera

exhibit these.

3. Undulating membrane - a protoplasmic membrane with a flagellar rim extending

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 out like a fin along the outer edge of the body of some flagellates. Moves in a
wave-like motion.

4. Costa - a thin, firm rod-like structure running along the base of the undulating
membrane in some flagellates.

5. Cytosome - a rudimentary mouth; also referred to as a gullet.

C. Identification of a flagellate is based upon:

1. Size
2. Shape
3. Motility
4. Number and morphology of nuclei
5. Number and location of flagellae
6. Location in the body of the host

D. Intestinal flagellates

1. Giardia lamblia - probably the first described protozoan pathogen of humans.

Giardia lamblia trophozoites G. lamblia cysts G. lamblia cyst (iodine)

a. Most common protozoan parasite in the U.S.A.

b. Life cycle - man ingests cysts from fecally contaminated environment;

the organism excysts in the upper intestine; trophozoites multiply and
attach to the intestinal mucosa, sometimes entering secretory tubes,
even the gall bladder. Trophozoites and cysts are passed in the feces.

c. diagnosis - identification of cysts or trophozoites in stool specimens or

duodenal contents. Irregular shedding pattern results in a showering of
organisms at times, while being difficult to detect at other times.

d. Morphology - very distinctive. Dorsal-ventrally flattened, and Bi-laterally

symmetrical.

1) Cyst - Measures 9 x 12 micrometers and contain 2 to 4 nuclei;

the karyosome is centrally located, with little or no peripheral
chromatin; parabasal bodies are present.

2) Trophozoite - Four pairs of flagella - one pair located anterior,

two pair located ventral, and one pair located posterior. An
axostyle and parabasal bodies are present. motility resembles a
falling leaf uses sucking discs to adhere to intestinal wall;
interferes with absorption of nutrients

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 e. Epidemiology - prevalence 1 to 30%, depending upon the population
surveyed; often occurs in epidemics, especially in childrens day care
centers; can be transmitted in water. Cysts remain viable as long as 3
months when protected from direct sunlight and excess heat; resistant to
chlorination. Sexual transmission has been well documented.

f. Pathology and Clinical Manifestations - symptoms can be severe;

diarrhea, foul - smelling, greasy, mucus-laden stools, flatulence, nausea,
cramps. Most infections are asymptomatic; chronic cases experience
weight loss, malabsorption of fat, protein, folic acid, and fat-soluble
vitamins.

2. Dientamoeba fragilis

Dientamoeba fragilis trophozoite D. fragilis trophozoite D. fragilis trophozoite

a. General - Until recently, classified as an amoeba; electron microscopy

and immunological studies have recently suggested a flagellate nature.

b. Laboratory diagnosis - detection of bi-nucleated (40% - 60%)

trophozoites with fragmented karyosomes, each karyosome consisting of
4 to 8 granules of chromatin.

c. Diagnostic stage - the trophozoite in feces. There is no cyst stage

known.

d. Morphology

1) 1 to 2 nuclei, with little or no peripheral chromatin

2) karyosome is divided into 4 to 8 distinct granules

3) may rarely ingest cells

e. Pathology and Clinical Manifestations - infection is usually asymptomatic;

can be associated with diarrhea, anorexia, abdominal pain.

f. Distribution - worldwide, 1 - 20% prevalence.

g. Association with the pinworm Enterobius vermicularis - it is thought that

the organism is transmitted from host to host within the egg of this
nematode.

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 2. Chilomastix mesnili

Chilomastix mesnili trophozoite C. mesnili cyst

a. A non-pathogen - must be differentiated from Giardia.

b. A commensal organism

c. Found in cecum and colon

d. Transmission - by ingestion of mature cysts.

e. Morphology

f. trophozoite

1) 4 flagella (3 extend anteriorly, and 1 is associated with the

cytostome); provide jerky directional movement
2) curved posterior
3) single nucleus - small eccentric karyosome, with granular
nuclear chromatin; the nucleus is always located anteriorly.
g. cyst

1) distinctive lemon shape

2) thick-walled with clear knob at one end
3) evidence of cytostome may be seen
4) 1 nucleus

4. The Trichomonads

a. Characteristics:

1) Undulating membrane - protoplasmic membrane with flagellar

rim extending out like a fin along outer edge of body. Moves in a
wave-like fashion.

2) Flagella - several in a tuft, provides locomotion

3) Axostyle - functions for support

4) Costa - firm rod-like structure running along base of the

undulating membrane.

5) Cytostome - rudimentary mouth

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 b. Trichomonas hominis a non-pathogen

Trichomonas hominis trophozoite

1) Commensal - must differentiate from pathogens

2) Transmission - direct person-to-person fecal transmission.
3) No cyst stage
4) Must differentiate from T. vaginalis - in instances where feces is
contaminated with urine.
5) Morphology - arc-shaped exhibiting a wobbly, jerky, yet
progressive motility

b.Trichomonas vaginalis

Trichomonas vaginalis trophozoites

1) Life cycle - trophozoite lives in the vagina, urethra, epididymis,

and prostate; multiplies via longitudinal fission; no cyst stage.
2) Mode of infection - sexual or fomites.
3) Diagnosis - ID of trophs in body fluids - wet mounts of
discharges or on PAP smears.
4) Pathology and Clinical Manifestations - females: vaginal
discharge; burning, Itching, or chafing. Frequency of urination or
dysuria. Relationship between trichomoniasis & cervical cancer.
Males: frequently asymptomatic. If the prostate is involved, the
patient may develop discharge, dysuria, and enlargement of
prostate with tenderness. There are reports of infections
(acquired during vaginal birth) of upper respiratory tract and
conjunctiva of newborns from infected mothers.

5) Morphology - exists only in trophozoite stage has an axostyle

and short undulating membrane that extends less than half the
body length; exhibits 4 flagellae, arranged in a tuft

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IV. Class Kinetofragminophora - The Ciliate

A. Balantidium coli

Balantidium coli trophozoite Balantidium coli trophozoite (unstained)

1. Epidemiology

Rarely found in USA. This is the only ciliate parasite of humans. It is prevalent in
tropical areas, or where poor sanitation, hygiene, and crowding occur. Increase
numbers of infections are expected in those with close, continuing contact with
swine.

2. Largest parasitic protozoan - the trophozoite is 30-120 x 25-125 microns; the

cyst averages 50 - 70 microns in diameter.

3. Pathology & Symptoms

a. Many infections are asymptomatic, with organism feeding on bacteria at

surface of mucosa.

b. Severe intestinal infections - with aid of hyaluronidase secretions,

organism burrows into submucosa, producing ulcers like those of E.
histolytica. Symptoms include dysentery, abdominal pain, nausea &
vomiting, fever, headache.

4. Life cycle - similar to that of E. histolytica.

a. The cyst is ingested via person-to-person transmission, or fecal

contamination in environment.

b. The cyst excysts in the small intestine

c. Trophozoites migrate to large intestine

5. Diagnosis

Diagnosed by observing cysts & trophs in fecal samples. Cysts are easily
missed cysts stain very dark with iodine, so the structures used in identification
(buccal cavity & macronucleus) are not always readily visible.

6. Morphology

a. Large, ovoid shape.

b. Contains two nuclei, 1 large kidney shaped (macronucleus) & 1 small

subspherical micronucleus adjacent to the macronucleus. Micronucleus
is difficult to see, if seen at all.

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 c. Body surface covered by spiral, longitudinal rows of cilia that provide
rotary, boring motility.

7. Disease names

a. balantidiasis
b. balantidial dysentery

8. Primary animal reservoir - pigs; monkeys probably of secondary importance.

V. Intestinal Coccidia
A. Introduction

1. A number of species parasitize humans: Isospora, Sarcocystis, Cryptosporidium,

& Toxoplasma

2. Have complex life cycles - most have 2-host life cycle.

3. Schizogony - asexual binary fission.

4. Sporogony -sexual reproduction

5. Diagnostic stages are often difficult to locate. They are easily overlooked due to
their nearly transparent appearance. Permanently stained smears not helpful.
Acid fast and giemsa stains are more often used. Oocysts do not stain with
iodine.

B. Sarcocystis

Sarcocystis spp. oocysts

1. Pathology - Sarcocystis bovihominis & S. suihominis are intestinal infections

similar to Isospora belli. Sarcocystis lindemanni is the name given to the
organism, which causes infection of the muscle.

2. Definitive host - humans. Pig (sui-) and cow (bovi-) are intermediate hosts.

3. Infective stage - ingestion of sarcocysts in meat (intestinal); ingestion of oocysts

from animal feces (muscle).

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 B. Isospora belli

Isospora belli oocyst (mature) Isospora belli oocyst (immature)

1. Definitive host - humans.

2. Schizogony and sporogony:

Schizogony - takes place upon initial infection when sporozoites escaping from
ingested oocysts invade the intestinal epithelium and multiply. The resulting
schizont releases trophozoites which invade other epithelial cells, and so the
infection progresses. Sporogony - sexual reproduction in which some of the
progeny of asexual reproduction initiate development into gametes (male &
female); microgametes migrate into lumen of bowel, and fertilize macrogametes
within epithelial cells; fertilized macrogametes develop into oocysts which are
passed as the infectious stage in the feces.

3. Diagnostic/infective stage

a. Immature oocysts - contain only one sporocyst, do not stain with iodine;
measure 12 x 30 microns.
b. Mature oocysts - contain two sporocysts, each of which contain four
sporozoites. Similarly, do not stain with iodine.

c. The sporozoites are released when the oocyst wall is digested away in
the small intestine, they then invade the epithelial cells.

d. They develop into trophozoites that multiply by binary fission

(schizogony) and are released when the infected epithelial cell ruptures.

e. This process can be repeated, or the organisms can undergo a

transformation into macrogametocytes & microgametocytes and initiate a
cycle of sexual reproduction.

4. Intermediate hosts - none.

5. Pathology - ranges from asymptomatic to acute, severe dysentery.

C. Cryptosporidium parvum

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 Cryptosporidium parvum oocysts
(acid-fast stain)
1. Site of infection - primarily an intestinal infection, cryptosporidiosis can become
systemic in AIDS patients. Organisms develop within the microvillous region of
epithelial cells lining the small intestine.

2. Originally considered an animal parasite (rodents, cattle and sheep), it is now

known that a wide variety of animals are naturally infected. Organisms from
some (fish, fowl and reptiles) are not believed to be able to infect humans.

3. Transmission - an endemic cycle is maintained via person-to-person, fecal-oral

route transmission. The parasite is a relatively common finding in day-care
diarrhea. It can be sexually transmitted. It exhibits a marked potential for being
waterborne due to significant resistance to disinfection. The potential for animal-
to-person transmission should not be underestimated.

4. Infections occur primarily in people with compromised immune systems. These

can be serious infections. It is readily accepted that the organism will readily
infect immunocompetent persons also. It is rapidly becoming a pediatric disease.

5. Pathology - although the condition can be asymptomatic, most infections cause

moderate to severe diarrhea of one to two weeks in the immunocompetent
patient. In the immunosuppressed patient, the condition is protracted and life
threatening. At this time, there is no drug effective against this parasite.

6. Identification - oocysts are 2 - 5 microns in diameter; do not stain with iodine;

and are acid-fast.

D. Cyclospora cayetanensis

Cyclospora cayetanensis oocysts Cyclospora cayetanensis oocyst Cyclospora cayetanensis oocyst

(acid-fast stain) (acid fast stain) (Variable acid fast reaction)

1. General - Relatively new to this country, this parasite has been detected in the
stools of infected individuals in the tropics for quite some time. The first outbreak
in the USA occurred in medical residents in Chicago in 1980, and a large multi-
state outbreak occurred the summer of 1996. During this outbreak, Texas had
more than 100 cases over a period of about six weeks, while no more than one
case had been reported during any previous year.

2. Animal reservoirs - not known. Other Cyclospora species are known to infect a
variety of animals, but C. cayetanensis is the name designated for the only one
known to infect humans (at this time).

3. Transmission - Contaminated food or water. The Chicago outbreak was

epidemiologically associated with drinking water; the summer 1996 outbreak was
tied to contaminated raspberries (imported from Central America) and possibly to
strawberries grown in California. Due to an extended time period (~10 - 14 days)

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 being required for oocyst sporulation, person-to-person spread is considered
unlikely.
4. Pathology - Infected individuals experience a diarrhea similar to that experienced
with Cryptosporidium infections. While some cases were less debilitating than
others, asymptomatic cases were not thought to have occurred. Trimethoprim-
sulfamethoxazole is an effective treatment in non-sulfa sensitive individuals.

5. Identification - Oocysts are spherical, 7 - 10 microns in diameter, and present as

variably acid-fast. Acid-fast procedures utilizing carbol-basic fuschin proved
superior in demonstrating this parasite.

E. Microsporidium

Microsporidium spp. spores Microsporidium spp. spores

1. General as of this time, infections are thought to be limited to AIDS patients.

2. Transmission person-to-person, via the fecal-oral route; can be sexually

transmitted during sex practices involving contact with feces.
3. Pathology - similar to that of Cryptosporidium infections.

4. Identification a very small organism, spores average 1.0 by 1.7 microns. They
stain a reddish-pink color with Chromotrope stain. Some stained spores exhibit a
dark staining belt across the middle of the organism.

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