MISCELLANEOUS  Cyst: 43-66 um in size; has 2 nuclei

(one kidney-shaped macronucleus,

PROTOZOA one small spherical micronucleus that
may NOT be observable); has a
double cyst wall.
General Characteristics

 Ciliates

– Motile by cilia – hairlike cytoplasmic

extensions

– Trophozoites and cysts are part of the

life cycle.

– Balantidium coli is the only species

pathogenic for humans.

 Select sporozoa

– Isospora belli
Laboratory Diagnosis of B. coli
– Cryptosporidium parvum
■ Stool Examination -For examining the
– Blastocystis hominis presence of trophozoites and cysts.

– Cyclospora cayetanensis ■ Sigmoidoscopy material examination

– Toxoplasma gondii ■ Permanent staining

■ Treatment – Metronidazole

Balantidium coli

 Causative agent of Isospora belli

balantidiasis/balantidial dysentery
 Causes isosporiasis, which is
characterized by mild diarrhea to
 Largest protozoan parasite
severe dysentery.

 only ciliate pathogenic for humans

 Transmission is by ingestion of the
infective oocyst in contaminated
 Transmission of the infective cyst is
food and water.
through contaminated (feces) water
or food.
 Humans are the definitive host; there
are no intermediate hosts.
 Trophozoite: 28-152 um in length, 22-
123 um wide; has 2 nuclei (one
 Oocyst: 25-35 um long, 10-15 um wide;
kidney-shaped macronucleus, one
transparent and oval in shape with a
small spherical micronucleus); has a
twolayered, colorless and smooth cell
rotary, boring motility.
wall.
 Sporocyst and sporoblast: 3. Flood slide with Acid Alcohol for 30
surrounded by a smooth, colorless, seconds. Rinse with water.
two-layered cell wall.
4. Counterstain by flooding the slide with
Methylene Blue for 30 seconds. Rinse with
water.

5. Dry the slide.

6. View organisms using the oil immersion

objective of your microscope

Laboratory Diagnosis of I. belli

Auramine-rhodamine staining
 Fresh feces and duodenal contents –
1. Prepare a thin smear of the specimen
specimen of choice for the recovery
on a sterile microscopic glass slide, and
of I. belli
gently heat fix the smear avoiding
 Enterotest or String test – for light
overheating.
infection
 Direct wet preparations – Iodine wet 2. Add enough quantity of the Auramine-
preparation Rhodamine Dyes (Flooding) on the smear
 Concentration or flotation techniques and allow it to stand for 15 minutes and
– Sheather’s sugar flotation ensure the dyes stain the smear well. Do
 Polyvinyl alcohol (PVA) preservation not apply heat.
 Acid-fast or auramine-rhodamine
3. Rinse the stained smear with water until
stains
no color appears in the effluent. Ensure
Sheather’s sugar flotation the water is chlorine-free water possibly
distilled water since chlorine interferes
■ use solutions (Sheather’s sugar) which
with fluorescence.
have higher specific gravity than the
organisms to be floated so that the 4. Add the decolorizing agent
organisms rise to the top and the debris (acidalcohol) for 2-3 minutes to destain
sinks to the bottom. The main advantage and wash thoroughly with distilled water
of this technique is to produce a cleaner and remove excess water by shaking the
material than the sedimentation slide.
technique. The disadvantages of most
5. Flood the smears with potassium
flotation techniques are that the walls of
permanganate (counterstain) for 2
eggs and cysts will often collapse, thus
minutes exactly. Note: long periods of
hindering identification. Also, some
counterstaining can quench the
parasite eggs do not float
fluorescence of the organism.
Acid-fast staining
6. Rinse thoroughly with distilled water
1. Air dry and heat fix a thin film of and allow to air dry. Do not blot.
microorganisms. Allow the slide to cool.
7. Examine under a fluorescent
2. Flood the slide with Carbol fuchsin. microscope.
Steam the slide with a Bunsen burner over
the sink. Let the slide set for 5 minutes.
Rinse with water.
 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. Blastocystis hominis

 In the immunosuppressed patient,  currently classified as an ameba, but

the parasite causes a wide range of rRNA analysis indicates it is related to
debilitating problems, including algae and water moulds.
malabsorption and stomach, liver,
and respiratory disorders.  Associated with diarrhea and
abdominal pain
 Transmission of the infected oocyst is
through contaminated food or water  Transmission is through contaminated
(rodent, cow, pig, or chicken feces). food and water.
Human-to-human transmission has
been documented in daycare  Morphology: The classic form varies in
centers. diameter from 4 to 60 um and
contains a large central body that fills
 Oocyst characteristics about 90% of the cell volume. There is
an outer ring of cytoplasm with
1) The oval oocyst ranges in size from 4 to several nuclei around the central
6 um. body.
2) Oocysts contain four sporozoites
enclosed within a thick cell wall.
 Diagnosis:
3) The cytoplasm may contain several  Microscopic examination of stool
dark granules. sample
 Iodine wet preparations
 Diagnosis:  Permanent stain preparations
 Microscopic detection of acid-fast  Stool culture
oocysts in stool or small bowel
mucosal epithelial cells – Boeck, Drohlav, Nelson & Jones media
 Enterotest
 Enzyme-linked immunosorbent assay
(ELISA)
 Indirect immunofluorescence (IIF)
 Zinc sulfate flotation/Sheather’s sugar
flotation
 Cyclospora cayetanensis ■ Congenital toxoplasmosis occurs in
premature or antibodydeficient infants,
 Humans are the only host for C.
where symptoms include splenomegaly,
cayetanensis.
jaundice, and fever. CNS infections can
 Nonbloody diarrhea is the most
lead to developmental complications,
common symptom, although
including vision and hearing problems,
infections can be asymptomatic.
hydrocephalus, and mental retardation.
 Oocyst: 7-10 um in diameter; has 2
sporocysts and each sporocyst ■ In patients with immunosuppression,
contains 2 sporozoites such as AIDS, the parasite becomes
 Sporocysts – infective stage localized in the CNS with symptoms of
encephalitis and brain lesions, often
 Diagnosis: resulting in death.
 Examination of stained fecal
smears. The oocysts will stain with
the modified Kinyoun's acid-fast Transmission to humans
stain.
 Flotation methods – Zinc sulfate ■ The sexual stage of reproduction occurs
flotation/Sheather’s sugar in the intestinal tract of house cats. The
flotation infective form (oocysts) of the parasite is
passed in the stool, and the ingestion of
cat feces-contaminated food and water
can produce infection.

■ Ingestion of undercooked meat (lamb

and pork) containing viable tissue cysts

■ Transplacental transmission from the

infected mother to the fetus.

Morphology

■ Tachyzoites (trophozoites) range in size

from 1 to 3 um and are crescent to round
Toxoplasma gondii
in shape.
■ Causes Toxoplasmosis, congenital
■ Cysts contain many bradyzoites.
toxoplasmosis, and cerebral
toxoplasmosis. ■ Because tachyzoites and bradyzoites
are small and no single organ is typically
■ Toxoplasmosis is characterized by a
involved, it is difficult to diagnose
broad spectrum of symptoms depending
infection by microscopic examination of
on the individual's state of health. T.
tissue samples.
gondii has a predilection for central
nervous system (CNS) infections.

■ 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.
 Laboratory Diagnosis

 Serologic testing for Toxoplasma

antibody:

 Sabin-Feldman Dye Test - Most

sensitive and specific test for
toxoplasmosis

 Double-sandwhich ELISA method -

Recommended test for the
determination of IgM antibodies in
congenital infections

 Indirect fluorescent antibody (IFA)

test – detects both IgM and IgG levels

 Indirect hemagglutination (IHA)

 ELISA

Sabin-Feldman Dye Test

 Principle: The test is based on the

presence of certain antibodies
that prevent methylene blue dye
from entering the cytoplasm of
Toxoplasma organisms.

 When live virulent tachyzoites of

Toxoplasma gondii are
incubated with normal serum,
tachyzoites become swollen and
stain deeply blue when
methylene blue is added.