P A R T 5 PROTOZOA

Learning Objectives
At the end of the topic, the students should be able to:

 Illustrate the general morphology of protozoa

 Explain the pathophysiology, life cycle, infective stages, modes of transmission, prevention and control
 Distinguish the diagnostic features of each parasite.
 Examine the different specimens used for identification
 Identify correctly the diagnostic features of each parasite.
 Perform the laboratory tests
 Discuss specimen collection, transport, processing, preservation and disposal

 INTESTINAL PROTOZOA

o General Characteristics

 Pseudopods are extensions of cytoplasm providing motility unique to amebae.

 Trophozoite andcyst stages are part of the amebae life cycle.
 Most amebic infections are spread to humans through contaminated water.
 Cyst is the infective stage, whereas the trophozoite is the active reproduction stage destroyed by stomach
acid.
 Laboratory identification: Microscopic identification of cysts (in formed stools) and trophozoites (in
liquid stools) based on size, nuclear characteristics, and inclusions
 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

o Intestinal Amebae

Entamoeba histolytica

 The only ameba pathogenic for the gastrointestinal tract

 Amebic colitis is characterized by abdominal cramping, anorexia, fatigue, and diarrhea.
 Amebic colitis can also cause ulcers and amebic dysentery.

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  Extraintestinal amebiasis primarily involves infections of the liver, but it is a rare complication.
 Additional conditions include infections of the spleen, brain, and lungs.
 Life cycle: Cysts are infective when ingested. Excystation occurs in the small intestines. Infective cysts are
passed in stools and are resistant to environmental 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 is fine 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 by means of pseudopods.
 E. histolytica trophozoites contain one nucleus, and they resemble those found in the cyst.
 Cytoplasm is finely granular and may contain red blood cell (RBC) inclusions.
 The presence of intracellular RBCs in intestinal amebae is considered diagnostic of E. histolytica.
 Morphologically, E. histolytica is identical to the nonpathogen E. dispar. These two species can be
differentiated by immunologic assays detecting surface antigens

Entamoeba coli

 E. coli is gener
 ally nonpathogenic but may cause intestinal problems in immunosuppressed patients.
 If found in a stool specimen, E. coli can indicate the presence of pathogenic organisms.
 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.

Blastocystis hominis

 B. hominis is currently classified as an ameba, but rRNA analysis indicates it is related to algae and water
moulds.
 Associated with diarrhea and abdominal pain

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  Transmission is through contaminated food and water.
 Diagnosis: Microscopic examination of stool sample
 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

 Entamoeba gingivalis: Causes asymptomatic mouth and genital tract

infections
 lodamoeba biitschlii: Nonpathogenic intestinal parasite
 Endolimax nana: Nonpathogenic intestinal parasite
 Entamoeba hartmanni: Nonpathogenic intestinal parasite

o Flagellates

o General characteristics

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

o Morphologic terms associated with flagellates

 Axostyle: Rodlike structure that functions in cellular support

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

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

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  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 vm long to 10(
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 are present.

Chilomastix mesnili

o Generally nonpathogenic but has been associated with disease in immunosuppressed patient
o Infection is acquired from contaminated food or water containing the cyst stage, which is infective.
o Diagnosis is by microscopic examination of stool samples.
o 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.
o 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.

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.

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  Most cells contain two nuclei without peripheral chromatin but with
clumps of nuclear chromatin.
 The cytoplasm is vacuolated with bacterial inclusions.

o Ciliates

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

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

o Morphology

 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 jam in width.
 Like the cyst, trophozoites contain two nuclei.
 Has one or two contractile vacuoles with cilia around the cell

 INTESTINAL SPOROZOA

A. 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 of debilitating problems, including
malabsoiption 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.

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 c) The cytoplasm may contain several dark granules.

B. 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.

C. 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.

 EXTRAINTESTINAL PROTOZOA

A. Plasmodium

1. Causative agents of malaria: P. vivax, P. falciparium, P. malariae, and P. ovale

2. Plasmodium spp. have two life cycle phases.

a. Sporogony: Sexual phase that occurs within the intestinal tract of the mosquito
b. Schizogony: Asexual phase that occurs in the human host

3. Transmission
a. Occurs with the bite of a female Anopheles mosquito that harbors the infective sporozoites in the salivary gland
b. Other forms of transmission include contaminated blood products, contaminated needles, and congenital malaria.

Diagnosis is made by clinical symptoms and microscopic examination of blood smears.

a. Because of the rapid progression of P. falciparum infections, examination of blood smears for malaria should be
considered a STAT procedure.
b. Ideally, blood should be collected by finger stick and blood smears made immediately. Alternatively,EDTA
(ethylenediaminetetraacetic acid) can be used as an anticoagulant in a venipuncture. Heparin can cause distortion of the
parasites.
c. Thick smears: A large drop of blood is placed on a slide and allowed to air dry. The RBCs are lysed in distilled water, and
the material is stained with Giemsa stain.
d. Thin smears: A drop of blood is placed on a glass microscope slide, and
the blood is spread out on the slide using another slide. The smear is fixed in methanol to prevent RBC lysis and then
stained.

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 e. Both thick and thin smears are thoroughly examined microscopically. The thick smear allows examination of about 20
times more blood volume than the thin smear, so it is much more sensitive. However, because the RBCs are intact, it is easier
to identify the parasites in thin smears, which makes them more specific.

Plasmodium morphology

a. Trophozoites or ring forms

1) Erythrocytic intracellular ringlike appearance
2) Giemsa or Wright's stain will show a blue cytoplasmic ring connected to a red chromatin dot.
3) Mature trophozoites will lose the ring appearance but will contain remnants of the cytoplasmic ring and chromatin dot.

b. Schizonts
1) Active chromatin activity causes the parasite to increase in size.
2) Pigmented granules are numerous and reddish-brown in color.
3) Schizonts contain merozoites; the number and arrangement depend
on the species.

c. Gametocytes
1) Characterized by a chromatin mass staining pink to purple.
2) The gametocytes of most species are round to oval in shape. P.falciparium gametocytes are "banana shaped."
3) Pigmentation varies by species.

Diagnosis is primarily made by microscopic examination of Giemsa (stain of choice) or Wright's stained smears.

Life cycle

a. The sporozoite is the infective stage transmitted to humans by Anopheles mosquitoes.

b. The sporozoites infect hepatic cells and begin the exoerythrocyte cycle. Plasmodium spp. undergo schizogony, an asexual
form of reproduction.
c. Following schizogony, merozoites are produced that invade erythrocytes.
d. A dormant form of P. vivax and P. ovale, called hypnozoites, can remain in the liver. Reactivation of the hypnozoites
results in a recurrence (true relapse).
e. Merozoites infect RBCs and initiate the erythrocyte cycle. Merozoites develop into ring forms and reproduce by
schizogony.Instead of undergoing schizogony, some merozoites develop into microgametocytes or macrogametocytes.
These stages are transmittedto the mosquito during human blood meal for completion of the life cycle (sexual phase).
g. The RBC form of the parasites can be nearly eliminated by treatment or an immune response, and the patient may become
asymptomatic. After several weeks, the parasites can increase in number and the symptoms will return; this is referred to as
recrudescence. All four Plasmodium spp. can cause a recrudescence.

Plasmodium vivax

a. Infected erythrocytes appear enlarged and pale with prominent Schuffner's dots. Only reticulocytes are infected, thus
limiting the parasitemia to 2-5
b. Trophozoite: Ring stage is one-third the size of an RBC; mature trophozoites fill the entire RBC.
c. Schizont contains 12 to 24 merozoites.
d. Gametocyte: Round to oval with a large chromatin mass that almost fills the RBC
e. Fever cycle lasts 48 hours.
P. vivax causes benign tertian malaria following a 10- to 17-day incubation period. It is the most common cause of malaria.

Plasmodium falciparum

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a. Infected erythrocytes appear normal in size, and all ages of RBCs can be infected, which can result in a large number of
infected cells.
b. Trophozoite: Ring stage is one-fifth the size of the RBC, and multiple rings are found in a single RBC. Some trophozoites
will have two chromatin dots in one ring form.
c. Schizonts are rarely seen in peripheral blood smears.
d. Crescent- or banana-shaped gametocytes are diagnostic of P. falciparium.
e. Miscellaneous characteristics: The interval between paroxysms (intense fever and chills) is 24 hours. Patients have a high
ratio of infected RBCs to uninfected RBCs compared to other Plasmodium spp. P. falciparum causes malignant tertian
malaria (blackwater fever) following a 7- to10-day incubation period.

Plasmodium malariae

a. Infected erythrocytes appear normal in size without dots; P. malariae prefers to infect older RBCs.
b. Trophozoites appear similar to P. vivax but stain a more intense blue. Mature trophozoites can produce band forms,
which spread across the diameter of the RBC.
c. Schizonts average 8 to 12 merozoites arranged in rosettes.
d. Gametocytes resemble P. vivax.
e. Fever cycle is 72 hours.
f. P. malariae causes quartan or malarial malaria following an 18- to 40-day incubation period.

Plasmodium ovale

a. Infected RBCs appear enlarged with thicker ring forms and contain Jame's dots. Infected RBCs resemble those infected
with P. vivax; however, P. ova/e-infected RBCs are often oval shaped and haveirregularly shaped membranes with
projections. Like P. vivax, only
reticulocytes are infected.
b. Trophozoites maintain their ring appearance as they develop.
c. Schizont: Averages 4 to 8 merozoites arranged in rosettes
d. Gametocyte resembles P. vivax but slightly smaller.
e. P.ovale causes benign tertian or ovale malaria following a 10- to 20-day

Babesia microti

1 . B. microti causes babesiosis, which can affect the spleen, liver, and kidneys. B. microti is an erythrocytic intracellular
parasite, like Plasmodium spp., that can also cause hemolytic anemia.
2. Babesiosis is a self-limiting infection; death is a rare outcome.
3. The infective sporozoite is transmitted to humans by a tick bite (Ixodes scapularis).
4. Diagnosis is made by blood smear examination and serologic testing. It is difficult to differentiate Babesia spp. from
Plasmodium spp.
5. Ring form characteristics
a. Size ranges from 3 to 5 um.
b. Cytoplasm: Minimal with two or more chromatin dots
c. Two to four rings per RBC are often seen, sometimes appearing like a "Maltese cross."

Toxoplasma gondii

Toxoplasmosis is characterized by a broad spectrum of symptoms depending on the individual's state of health. T. gondii
has a predilection for central nervous system (CNS) infections.
a. In healthy individuals, toxoplasmosis often resembles infectious mononucleosis and produces fatigue, swollen lymph
glands, fever, and myalgia. The disease can become chronic and affect the heart and liver.

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 Congenital toxoplasmosis occurs in premature or antibody-deficient infants, where symptoms include splenomegaly,
jaundice, and fever. CNS infections can lead to developmental complications, includingvision and hearing problems,
hydrocephalus, and mental retardation.
c. In patients with immunosuppression, such as AIDS, the parasite becomes localized in the CNS with symptoms of
encephalitis and brain lesions, often resulting in death.

Transmission to humans
a. The sexual stage of reproduction occurs in the intestinaltract of house cats. The infective form (oocysts) of the parasite is
passed in the stool, and the ingestion of cat feces-contaminated food and water can produce infection.
b. Ingestion of undercooked meat (lamb and pork) containing viable tissue cysts
c. Transplacental transmission from the infected mother to the fetus

Diagnosis: Serologic testing for Toxoplasma antibody

Because tachyzoites and bradyzoites are small and no single organ is typically involved, it is difficult to diagnose infection
by microscopic examination of tissue samples.

Tachyzoites (trophozoites) range in size from 1 to 3 um and are crescent to round in shape.

Cysts contain many bradyzoites.

Naegleri fowleri

Causes amebic meningoencephalitis, which is often fatal within 3-6 day

N.fowleri is found in lakes, ponds, and swimming pools where the water is warm.

Life cycle: Trophozoites are the infective stage. N. fowleri does not need a host to survive and can be free living, spending its
entire life cycle in the external environment.

The amebae are contracted from contaminated water, where trophozoites enter the body through the nasal mucosa and
migrate along the olfactory nerve to the brain.

Diagnosis is made by finding the organism (ameba) in cerebrospinal fluid (CSF) or brain biopsies.

Morphology

a. Cyst characteristics
1) The round cyst ranges in size from 10 to 13 urn.
2) N.fowleri cysts contain a single nucleus, without peripheral chromatin.
3) The karyosome is centrally located and large.

b. Trophozoite characteristics
1) Size ranges from 10 to 23 jam, and they are motile by means of blunt pseudopods.
2) Trophozoites contain a single nucleus, without peripheral chromatin.
3) The cytoplasm is granular and vacuolated.

c. Flagellate characteristics
1) Flagellates range in size from 7 to 15 jam and are pear shaped. They are motile by means of two flagella.
2) The single nucleus is indented.

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3) Karyosome is centrally located and large.
4) The cytoplasm is granular and vacuolated.

Acanthamoeba

1. Causes amebic encephalitis and amebic keratitis (cornea infection)

2. The life cycle is not well characterized.
a. The eye is directly invaded by trophozoites, producing keratitis.
b. Skin, respiratory tract, and CNS infections are caused by the cyst or trophozoite stage (unknown entry route).

Diagnosis is made by finding the cyst or trophozoite stages in CSF.

Morphology

Cyst characteristics
1) Size ranges from 8 to 25 um with a jagged edge and a round shape.
2) Single nucleus without peripheral chromatin
3) The karyosome is centrally located and large.
4) The cytoplasm is granular and vacuolated.

Trophozoite characteristics
1) Size ranges from 15 to 45 um; motility is by spinelike pseudopods.
2) Contains a single nucleus without peripheral chromatin
3) The karyosome is centrally located and large.
4) The cytoplasm is granular and vacuolated.

Trichomonas vaginalis

1. Causes vaginitis in women, whereas men are generally asymptomatic carriers

2. T. vaginalis is a sexually transmitted disease and can infect neonates (aspiration pneumonia) during delivery.

3. Trophozoites are the infective stage and infect the epithelial or mucosal lining of the vagina, urethra, and prostate gland. T.
vaginalis does not have a cyst stage.

4. Diagnosis: Trophozoites are usually detected during a microscopic urinalysis.

5. Trophozoite characteristics
a. Trophozoites average about 30 um in length. They are motile, with an undulating membrane, and are pear shaped. See
Figure 8-7 •.
b. Single prominent nucleus
c. Flagella: Three to five anterior and one posterior
d. Large axostyle with cytoplasmic granules

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 Hemoflagellates

1. General characteristics
a. Hemoflagellates inhabit the blood and tissues of humans.
b. Four stages of development: amastigote, promastigote, epimastigote, and trypomastigote

1) Amastigote: Nonflagellated oval form, found in tissue

2) Promastigote: Flagellated stage found in the vector, rarely seen in the blood

3) Epimastigote: Long, slender flagellated form found in arthropod vectors

4) Trypomastigote: Has an undulating membrane running the length of the body; found both in the vector and bloodstream
of humans. See

Trypansoma brucei

a) T. brucei causes African trypanosomiasis or sleeping sickness, and infection affects the lymphatic system and CNS.
Swollen lymph nodes at the posterior base of the neck (Winterbottom's sign) are sometimes present.
b) Subspecies gambiense andrhodesiense are named according to their geographic location.

Trypansoma cruzi

a) Causes Chagas disease or American trypanosomiasis, which is characterized by lesion formation (chagoma),
conjunctivitis, edema of the face and legs, and heart muscle involvement leading to myocarditis.
b) Mostly found in South America

3. Leishmania

a. Human leishmaniasis
1) Cutaneous leishmaniasis is characterized by skin and mucousmembrane ulcers.
2) Disseminated leishmaniasis: Liver, spleen, and reticuloendothelial involvement

b. Diagnosis: Finding the amastigote in the blood or tissue and serologic testing

TRUST THE PROCESS

GOD BLESS YOU!

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