Topic 2: PHYLUM PROTOZOA (Intestinal Protozoans)

Activating Prior Knowledge

Intended Learning Objectives

At the end of the topic, the students should be able to:
1. Illustrate the general morphology of intestinal protozoans
2. Explain the pathophysiology, life cycle, infective stages, modes of
transmission, prevention and control
3. Distinguish the diagnostic features of intestinal protozoans
4. Examine the different specimens used for identification of intestinal
protozoans
5. Identify correctly the diagnostic features of each intestinal protozoan
6. Perform the laboratory tests
7. Discuss specimen collection, transport, processing, preservation and
disposal

Presentation of Contents

1. General characteristics of protozoan parasites

2. Classes of protozoan parasites
3. Different species of amoeba parasites discussing their life cycle, pathogenesis,
signs and symptoms of infection, treatment and management as well as prevention
and control
4. Laboratory diagnosisto evaluate for amoeba infections

INTESTINAL PROTOZOA

General Characteristics

1. Pseudopods are extensions of cytoplasm providing motility unique to

amoebae.
2. Trophozoite and cyst stages are part of the amoebae life cycle.
3. Most amebic infections are spread to humans through contaminated water.
 4. Cyst is the infective stage, whereas the trophozoite is the active
reproduction stage destroyed by stomach acid.
5. Laboratory identification: Microscopic identification of cysts (in formed
stools) and trophozoites (in liquid stools) based on size, nuclear
characteristics, and inclusions
6. Size is one of the most important criteria for identification.

Morphologic terms associated with protozoa

Karyosome- area of chromatin within the nucleus
 Peripheral chromatin - nucleic acid combined with protein found
along the nuclear membrane
Excystation - development of a cyst into a trophozoite
Encystation - development of a trophozoite into a cyst
Chromatoid bar- Rod-shaped, RNA containing structure found in the
cytoplasm

Intestinal Amebae

1. Entamoeba histolytica

1. The only ameba pathogenic for the gastrointestinal tract

2. Amebic colitis is characterized by abdominal cramping,
anorexia, fatigue, and diarrhea.
3. Amebic colitis can also cause ulcers and amebic dysentery.
4. Extra-intestinal amebiasis primarily involves infections of
the liver, but it is a rare complication.
5. Additional conditions include infections of the spleen, brain,
and lungs.
6. Life cycle: Cysts are infective when ingested. Excystation
occurs in the small intestines. Infective cysts are passed in
stools and are resistant toenvironmental stress.

o Morphology

 Cyst characteristics
 Cysts range in size from 8 to 22 um, and they are
spherical.
 E. histolytica contains one to four nuclei; peripheral
chromatin isfine and uniformly distributed.
 The karyosome is centrally located.
 Cytoplasm is finely granular with chromatoid bars
with round ends.
  Trophozoite characteristics

 Trophozoites range in size from 5 to 70 cm,and they

are motile bymeans of pseudopods.
 E. histolytica trophozoites contain one nucleus, and
they resemblethose found in the cyst.
 Cytoplasm is finely granular and may contain red
blood cell (RBC)inclusions.
 The presence of intracellular RBCs in intestinal
amebaeis considered diagnostic of E. histolytica.
 Morphologically, E. histolytica is identical to the
nonpathogenE. dispar. These two species can be
differentiated by immunologicassays detecting
surface antigens

Entamoeba coli

1.E. coli is generally nonpathogenic but may cause intestinal

problems in immunosuppressed patients.
2. If found in a stool specimen, E. coli can indicate the presence of
pathogenic organisms.
3. Needs to be differentiated from E. histolytica for purposes of
treatment

o Morphology

 Cyst characteristics
 Cysts range in size from 8 to 40 cm and they are
spherical.
 E. coli contains one to eight nuclei; the peripheral
chromatin is coarse and unevenly distributed.
 Young cysts may contain a large central glycogen
mass pushing two nuclei to the periphery of the cell.
 The karyosome is eccentric and large.
 The cytoplasm is coarse with thin
chromatoid bars with pointed ends.
 Trophozoite characteristics

 Trophozoites range in size from 10 to 60 cm, and they are

motile by means of short/blunt pseudopods.
 E. coli trophozoites contain a single nucleus with coarse,
unevenly distributed chromatin, and they resemble those
found in the cyst.
 The cytoplasm is coarse and vacuolated, with bacterial
 inclusions.

3. Blastocystis hominis

1. B. hominis is currently classified as an ameba, but rRNA

analysis indicates it is related to algae and water moulds.
2. Associated with diarrhea and abdominal pain
3. Transmission is through contaminated food and water.
4. Diagnosis: Microscopic examination of stool sample
5. Morphology: The classic form varies in diameter from 4 to
60 |om and contains a large central body that fills about 90%
of the cell volume. There is an outer ring of cytoplasm with
several nuclei around the central body.

o Other intestinal amebae

1. Entamoeba gingivalis: Causes asymptomatic mouth and

genital tract
infections
2. lodamoeba biitschlii: Nonpathogenic intestinal parasite
3. Endolimax nana: Nonpathogenic intestinal parasite
4. Entamoeba hartmanni: Nonpathogenic intestinal parasite

Intestinal Flagellates

General characteristics

1. Flagellates are a subclass of protozoa that have one or more

flagellum that provide motility.
2. All flagellates have a trophozoite stage, but several lack the
cyst stage.
3. Many flagellates live in the small intestines.
4. Giardia lamblia is the only pathogenic flagellate; it causes
mild to moderate diarrhea
5. Severe infections can lead to malabsorption.
6. Diagnosis is by microscopic examination of stool for
trophozoites or cysts.

o Morphologic terms associated with flagellates

1. Axostyle: Rodlike structure that functions in cellular support

2. Axoneme: The intracellular portion of the flagellum
 3. Undulating membrane: Flagellum finlike structure that
generates a wavelike motion
4. Cytostome: A rudimentary oral cavity

1. Giardia lamblia

o Taxonomy: G. duodenalis and G. intestinalis are synonyms.

o G. lamblia causes giardiasis (a form of traveler's diarrhea)
characterized by acute diarrhea, abdominal pain, and weight loss.
Self-limiting infections last 10-15 days, following a 10- to 35-day
incubation period.
o Infection is due to exposure to contaminated water and food (mostly
from wild animal stool).
o Campers and hunters are prone to infection after drinking untreated
water from streams.
 Cysts are the infective stage.
 Cysts pass through the stomach and excyst in the duodenum.
 Trophozoites attach to the duodenum mucosa.
 Encystation occurs in the large intestines, and the cysts will
pass in the stool.

o Diagnosis
 Microscopic examination of stool samples for trophozoites
and cysts
 Other diagnostic tests include the EnteroTest and antigen
detection by immunological assays (ELISA, etc.).

o Morphology

 Cyst characteristics
 G. lamblia cysts are oval shaped, and the average
size ranges from 12 um long to 8 um wide.
 Cysts contain four nuclei with no peripheral
chromatin.
 Cytoplasm is retracted from the cyst wall and may
contain two to four comma-shaped, median bodies.

 Trophozoite characteristics
 G. lamblia trophozoites have an average size of 15
um long to 10um wide.
 They are motile and pear shaped, with bilateral
symmetry and
two large nuclei on each side of a central axostyle.
  Trophozoites contain two oval-shaped nuclei,
without peripheral
chromatin.
 Trophozoites possess four pair of flagella.
 Two median bodies, two axonemes, and a sucking
disk arepresent.

2. Chilomastix mesnili

1. Generally nonpathogenic but has been associated with disease in

immunosuppressed patient
2. Infection is acquired from contaminated food or water
containing the cyst stage, which is infective.
3. Diagnosis is by microscopic examination of stool samples.

Cyst characteristics
 The cyst ranges in size from 5 to 10 jam in length and is
oval
shaped.
 C. mesnili contains a single nucleus without peripheral
chromatin.
 The karyosome is large and centrally located.
 The cytostome is well defined.

Trophozoite characteristics
 Size ranges from 5 to 25 jam in length and 5 to 10 um in
width; they are pear shaped and motile.
 Single nucleus without peripheral chromatin
 Karyosome: Eccentric and small
 Flagella: Three anterior and one posterior
 Cytostome is very large, and a spiral groove is present.

3. Dientamoeba fragilis

o Causes diarrhea, abdominal pain, and anal pruritus (itching)

o Many cases of diarrhea caused by D.fragilis occur in individuals
living in close quarters, such as inmates, college students, and
military recruits.
o D.fragilis infects the mucosal lining of the large intestines. There is
no cyst stage, and the life cycle is not well denned.
o Diagnosis is made from microscopic examination of trophozoites in
the stool. Multiple samples are required. The parasite is very
delicate
and stains poorly.
 o Trophozoite characteristics
 Size ranges from 5 to 19 um; they are motile by means of
hyaline pseudopods and are round shaped.
 Most cells contain two nuclei without peripheral chromatin
but with
clumps of nuclear chromatin.
 The cytoplasm is vacuolated with bacterial inclusions.

Intestinal Ciliate

o General characteristics
1. Motile by cilia
2. Trophozoites and cysts are part of the life cycle.
3. Balantidium coli is the only species pathogenic for humans.

1. Balantidium coli

 Causes balantidiasis, characterized by diarrhea to dysentery

 Transmission of the infective cyst is through contaminated
(feces) water or food

o Diagnosis: Microscopic examination of stool for cysts or

trophozoites

Cyst characteristics
 Ranges in size from 43 to 65 um and is round in
shape
 B. coli contains two nuclei; one, the macronucleus, is
kidney-shaped and very large. The micronucleus is
round and much smaller; it is rarely seen.
 Has a double cyst cell wall with numerous cilia
between the two cell walls
Trophozoite characteristics
 Trophozoites range in size up to 100 um in length
and 70 um in width.
 Like the cyst, trophozoites contain two nuclei.
 Has one or two contractile vacuoles with cilia around
the cell

Intestinal Sporozoans
1. Cryptosporidium parvum

 Causes cryptosporidiosis, which is characterized by

moderate to severe
diarrhea
 In patients with acquired immunodeficiency syndrome
(AIDS),
 Cryptosporidium infections are an important cause of death
due to dehydration.
 In the immunosuppressed patient, the parasite causes a wide
range ofdebilitating problems, including malabsorption and
stomach, liver, and respiratory disorders.
 Transmission of the infected oocyst is through contaminated
food or water (rodent, cow, pig, or chicken feces). Human-
to-human transmission has been documented in daycare
centers.
 Diagnosis: Microscopic detection of acid-fast oocysts in
stool or small bowel mucosal epithelial cells
 Oocyst characteristics:
a) The oval oocyst ranges in size from 4 to 6 jam.
b) Oocysts contain four sporozoites enclosed within a thick
cell wall.
c) The cytoplasm may contain several dark granules.

2. Cyclospora cayetanensis

 Humans are the only host for C. cayetanensis.

 Nonbloody diarrhea is the most common symptom, although
infections
 can be asymptomatic.
 Diagnosis is made by examination of stained fecal smears.
The oocysts will stain with the modified Kinyoun's acid-fast
stain and are 8-10 jam in diameter.

3. Isospora belli

 Causes isosporiasis, which is characterized by mild diarrhea

to severe dysentery
 Transmission is by ingestion of the infective oocyst in
contaminated food and water.
 Humans are the definitive host; there are no intermediate
hosts.
 Diagnosis: Microscopic examination of stool for oocysts by
wet mounts and/or acid-fas or auromine-rhodamine stains
  Oocyst characteristics:

1) The oval oocyst ranges in size from 25 to 40 um in

length.
2) The cytoplasm is granular and contains two sporoblasts
that contain four sporozoites each.

Application

Based on your readings of the lecture, please answer the following questions.
Kindly write your answers in your activity notebook.

1. What are the difference between:

a. Entamoeba histolytica and Entamoeba dispar?

b. Entamoeba coli and Entamoeba polecki?

2. Why are Entamoeba species regarded as the true amoeba parasites?
3. Discuss the life cycle of amoebic protozans? Which of the life cycle stages
are known as intermediate stages?
4. What do you think is the importance of:

a. encystation

b. excystation

c. binary fission

d. serologic diagnosis of protozoan infections

Feedback

Where there babies you know have suffered from diarrhea and who were brought
 by their mothers in a hospital? For sure, the attending physician requested for fecal
analysis as he/ she is suspecting of amoebiasis. Yes, the doctor maybe right but in
order to confirm that it is really an intestinal protozoa that causes the infection, a
laboratory technologist must confirm it since diarrhea can also be caused by
bacterias and viruses. Is it not that after a diagnosis of a protozoal infection, the
child was already given a medication and we know that the patient was cured after
the diarrhea and fever he used to suffer from was already managed? That is how it
works. So now you realize that indeed, the basis for treatment and management
starts from the diagnosis of a medical laboratory practitioner.

Interview a health worker, either a doctor, nurse, midwife or a medical

technologist and ask them about the following:

1. How do they rule out “amoebiasis”?

2. Aside from amobiasis, what other protozoan infection have they encountered
from their patients? How did they come to know about the diagnosis?
3. Ask how important the role of a medical technologist is in the diagnosis of
protozoan infections?

Summary
Intestinal protozoan infections is very common among infants and school- aged children
although there are also reports of cases from among adults and elderly people. The disease is
very easy to transmit through ingestion of fecal- contaminated food and water and all the more
that it is a concern because most cases are assymptomatic, meaning, they can transmit the
infection without knowing that they have the condition since the signs and symptoms are not
evident. The parasitic condition can cause severe dehydration and consequently death if not
detected early and should cases continue to suffer from severe diarrhea.
The diagnosis of any of the intestinal protozoan is challenging much as they are relentlessly
microscopic and have similar characteristics. A laboratory technologist must therefore be taught
of the salient features of each of the intestinal protozoan so as to provide himself of a clue for a
presumptive diagnosis.

Today, there are also rampant reports of Metronidazole- resistant cases which primarily is rooted
from the concern of misdiagnosis and eventually misuse of drugs for treatment. In which case,
focus must be given on diagnosis.
 Reflection
Activity 1

1. Fill in the table (differences between the intestinal protozoans):

Parasite Description of the Description of the Description of Unique
Trophozoite Stage Cyst Stage motility and feature/s of
motility the parasite
apparatuse
Entamoeba
histolytica
Entamoeba coli
Endolimax nana
Iodamoeba
butschlii
Gardia lamblia
Chilmastix
mesnili
Blastocystis
hominis
Balantidium coli
Isospora bellii

2. Discuss the following:

a. Why is Gardia lamblia cyst the most resistant cyst?

b. What is the importance of the Entero-capsule technique in the diagnosis of Gardia lamblia?
Enumerate its procedures.

c. Why are Cryptosporidium and Isospora species associated to AIDS patients?

d. What are the different forms of Blastocystis hominis? Which form is associated with
intestinal protozoan infection?
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ASCP Press, 2007.

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3. Boqitsh, Burton, Clint Carter and Thomas Oeltman. Human

Parasitology 3rded. USA: academic Press, 2005.

4. Garcia, Lynn Shore. Diagnostic Medical Parasitology 5th ed. USA:

ASM Press, 2006.

5. Heela, Judith and Frances Ingersoll. Essentials of Human Parasitology. USA: Delmar
Learning, 2001.

6. Ichhpujani R.L, Bhatia. Medical Parasitology, India: Jaypee Brothers

Medical Publisher, New Delhi, India, 2002.

7. Mcpherson, Richard A. and Matthew R. Pincus. Henry’s Clinical Diagnosis and

Management by Laboratory Methods 21st ed. Philadelphia: Elsevire Inc., 2007.

7. Peters, Wallace and Geoffrey Pasvol. Atlas of Tropical Medicine and

Parasitology 6th ed. Edinburg: Mosby, 2006.