PROTOZOA

OBJECTIVES

• Describe the general morphology of Protozoa.

• Explain the pathophysiology, life cycle, infective stages,

modes of transmission, prevention and control.

• Discuss the different laboratory test for the different

Medical Protozoa.

• Describe the diagnostic features of each parasite.

 MEDICALLY IMPORTANT TERMS

PSEUDOPODS - temporary arm/feet projection developed in

the direction of movement

CILIA - small hair-like structure responsible for locomotion

FLAGELLATE - whip-like appendages

TROPHOZOITE - vegetative and motile stage of of protozoa

CYST - non-motile and resistant stage of protozoa

SYNGAMY - sexual union or conjugation of two cells

ENCYSTATION - development of cystic stage from trophozoite in

unfavorable condition
 INTRODUCTION

• The Phylum Protozoa is classified into four subdivisions according to the

methods of locomotion:
1. Amoeba (Sarcodina) - pseudopodia

2. Flagellates (mastigophore - flagellate

3. Cilliates (Cilliata) - cilia

4. Sporozoans (Sporozoa) - no organ of locomotion

 GENERAL

• 45,000 protozoan species (8,000 species are parasitic, 25 species are

important to humans)

• Laboratory Diagnosis

• Transmission - human to human

• Diagnostic features:
nuclear structure
size
cytoplasmic inclusions
appearance of cytoplasm
 Protozoa

- unicellular parasites/single-celled organism

varies in size, shape, locomotor apparatus,
organelles,appearance of nucleus

Stages of development: trophozoites,cyst

*** in some protozoa ---- schizont, gametocytes,
bradyzoites, tachyzoites, sporozoites, oocyst

Methods of reproduction: Asexual or simple fission,

Sexual union of two cells (syngamy)
 AMOEBA
Genera of Amoeba:
Entamoeba,Endolimax,Iodamoeba

Stages of development: cyst and trophozoite

•Entamoeba gingivalis and Dientamoeba
fragilis- no cystic stage

Organ for locomotion: pseudopodia

 Entamoeba histolytica

Two forms of Entamoeba histolytica:

1. E. histolytica ( magma form) – large race,
pathogenic,hematophagous and tissue invading
2.E. hartmanni (minuta form) – small race and non
pathogenic
Trophozoite:clean- looking with one nucleus,ingested RBC
(pathognomonic)
Movement: progressive, directional
Cyst: round,1-4 nuclei,sausage shaped or cigar
shaped chromatoidal barr
 Pathology

Infective stage: quadrinucleated/mature cyst

*** Elaborate the enzyme HYALURONIDASE –

destroy the affected tissues

*** lesions produced by E. histolytica are

primarily intestinal and secondary extra-intestinal
***flask-shaped cavity or ulcer containing cytolyzed
cells, mucus and amoeba
The pathology of amoebiasis maybe classified into:
A. Asymptomatic amoebiasis
*patient---- carrier--- lesion is localized in the cecal area
B. Acute symptomatic amoebiasis
1. Amoebic diarrhea----numerous extensive ulcers, stools
are soft,diarrheic,defecation 3-6x a day
2. Acute amoebic dysentery----marked ulcerations in the
cecal-sigmoidal rectal regions severe diarrhea (6 – 8
X/day), stool with blood and mucus, painful defecation
C. Chronic amoebic dysentery
thickening of the colonic mucosa of the sigmoid and
cecal area forming a tumor-like mass called
“amoebomas”

D.Extra-intestinal amoebiasis
Hepatic amoebiasis,Pulmonary amoebiasis,Cerebral
amoebiasis,Amoebic pericarditis,Cutaneous
amoebiasis,Genital amoebiasis
Diagnosis

Intestinal amoebiasis
1. Stool
Microscopic examination
a.DFS
Solutions DFS:
*Quensel's solution – best for trophozoite
*Methylene blue
*Lugol’s iodine – for cyst
*Di Antonis – for cyst
b.Concentration techniques like FECT, MIFC and ZSFT
Cultivation:
Culture media for cultivation of amoeba:
a. LES – Locke’s Egg Serum
b. Boeck’s and Drbohlav’s
c. RES – Rice Egg Saline
d. Nutrient Agar Saline
e. Diamond
2. Serum
Serological tests: ELISA, indirect hemeagglutination
test (IHA), gel-diffusion precipitin (GDP) and indirect
immunofluorescence (IIF).
3. Examination of rectal biopsy for parasites
4. For extra-intestinal amoebiasis
liver aspirate – examined for the presence of parasite
liver scan,serological tests
 Other Amoeba

Entamoeba coli
Trophozoite:dirty looking – numerous food and
bacterial vacuoles
Movement:slow,sluggish
Cyst: round,1-8 nuclei,chromatoidal barr with splintered
ends (broomstick in appearance)
Endolimax nana – cross eyed cyst
Iodamoeba butschlii- large glycogen vacuole –
- Iodine cyst
Entamoeba gingivalis – trophozoite – multiple
pseudopodia,lyzed WBC,epithelial cells
Dientamoeba fragilis- with small round trophozoite,
Psudopodia are blunt and leaflike
Endolimax nana
Iodamoeba butschlii
Dientamoeba
Free-living Pathogenic Amoeba

Naegleria
Naegleria fowleri- pathogenic specie
Two morphologic forms of trophic stage:
amoeboid = with blunt pseudopodia and a vesicular
nucleus
flagellated = elongated and with two long, equal and
anteriorly located flagella
Cyst - round with double, smooth cyst wall wherein the
outer wall is perforated by 3 – 8 pores (ostioles),
single nucleus and spherical chromatoidal barr.
Pathology: PAM – Primary Amoebic
Meningoencephalitis
Naegleria may enter the nose of human
host while swimming in contaminated
water. From the nasal passages the
trophozoite migrate along olfactory
nerves, meninges and brain.
Acanthamoeba
Trophozoite:with a single vesicular nucleus and large
karyosome;pseudopodia are broad, anterior from
which slender, hyaline projections (acanthopods)
Cyst:Uninucleated and double-walled,the outer
ectocyst is wrinkled
Pathology: GAE- Granulomatous amebic encephalitis
Amebic or Acanthamoeba keratitis
• The life cycle of Acanthamoeba is not well
defined. It is presumed that most
infections invade CNS from the lower
respiratory tract or skin or some inhaled
the infective cyst from contaminated dust.
A rapid transformation from cyst to
trophozoite occurs in the nasal mucosa.
Direct invasion of the eye also occur.
 Atrial or lumen flagellates
Giardia lamblia
Trophozoite:
•pear-shaped or teardrop shaped
•prominent, ovoid, concave-sucking disk
•four pairs of flagella (8 flagella)
•two nuclei with a large karyosome
•axostyle made up of two axonemes
•sausage shaped parabasal body or median body
Movement:jerky, falling leaf
 Cyst:
•Ovoidal
• two to four nuclei
•axostyle and other fibrillar remnants of the organelles

Pathology:
Attachment of the parasites to the intestinal mucosa
provokes intense inflammation that result to secretions of
abundant mucus---general malabsorption of fats and
carbohydrates.
Chilomastix mesnili
Trophozoite:
•pear-shaped
•spiral groove
•three free anterior flagella (two short, one long) and a
delicate flagellum lying within the cystostome
•numerous food vacuoles.
Movement:stiff-rotary fashion (corkscrew motion).
Cyst:lemon-shaped, rounded at one end and conical at
the other end with a knob-like protuberance at the
anterior portion (nipple-shaped cyst)
Trichomonas species
General features:
No cystic stage, only the trophic stage is known.
Trophozoite:Pyriform with a curved,rigid axostyle,small
cytostome,the nucleus is spherical,blepharoplast is
present between the nucleus and the anterior margin
from which arises 3 – 5 flagella and a marginal
flagellum on an undulating membrane, the base
attachment being a heavy costa.

Movement of trophozoite:jerky,tumbling
Diagnosis:
Specimens:female patients:urine, urethral secretion,
vaginal and cervical secretions
Male patients: semen and prostatic secretions
a.Unstained wet mount-trophozoite
b. stain for permanent smear:Giemsa, Pap’s,
Romanowsky,Acridine Orange
Cultivation:Culture media:
Diamond’s modified medium or Feinberg and
Whittington culture
Basis for T. vaginalis T. hominis T. Tenax
differentiation
1. Size 15 – 20 microns 7 – 13 microns 5 – 12 microns

2. Siderophil present absent absent

granules
BLOOD AND TISSUE FLAGELLATES
Trypanosomes
Trypanosoma brucei gambiense
Disease : West African Sleeping sickness
Vector : Glossina specie (Tsetse fly)
Trypanosoma brucei rhodesiense
Disease : East African Sleeping sicknes
Vector : Glossina specie (Tsetse fly)
Trypanosoma cruzi
Disease : Chaga’s disease or American
trypanomiasis
Vector :Triatoma or Reduviid bugs (kissing bug or
Assassin bug)
 B. Leishmania

Leishmania tropica
Disease:Oriental sore or Old World cutaneous
Leishmaniasis (OWCL)
Vector : Phlebotomous specie (sandfly)
Leishmania braziliense
Disease: a.Espundia or muctaneous leishmaniasis (MCL)
b. Chiclero’s ulcer or New World Cutaneous
leishmaniasis (NWCL)
Leishmania donovani
Disease: Kala azar or visceral leishmaniasis
Vector :Phlebotomous specie (sandfly)
Stages of Development of Hemoflagellates

1.Amastigote or leishmania form – ovoidal without

free flagellum
2.Promastigote or Leptomonas form – elongated and
spindle-shaped with a free flagellum
3. Epimastigote or Crithidia – elongated and spindle-
shaped with free flagellum continous from the
anterior end backward along the margin of the
undulating membrane
4.Trypomastigote or Trypanosoma – the flagellum on
the margin of the undulating membrane ends at the
kinetoplast which is situated anterior to the nucleus.

Genus Leishmania---amastigote and promastigote

Trypanosoma brucei group---epimastigote and

Trypomastigote
Trypanosoma cruzi---all stages of development are
present
Diagnosis:
A. Trypanosomes
T. brucei group- stained blood preparation and lymph
aspirates-trypanosomes
T. cruzi--thick blood smear----trypanosomes
Xenodiagnosis---bugs
Serologic tests-- immunofluorescent antibody test,
indirect hemeagglutination assay
B. Leishmania
Specimen:Smear fromlesion
1. Stain-- Wright or Giemsa
2. 2. Cultivation - NNN (Nicole, Novy, Mc Neal)
3. Serological tests-CFT, FAT,counter-current
electrophoresis
4. Montenegro skin test
5. Formol-gel test making use of 0.1-ml serum and one
drop of formalin and the positive result is gel
formation---- screening test
Balantidium coli
Trophozoite:
•Ovoid or shape like a sac
•with spiral longitudinal rows of cilia
•with cytostome or primitive mouth
•With cytopyge and excretory pore
•two contractile vacuoles
•two nuclei, a large bean-shaped or kidney-shaped
macronucleus and a small spherical micronucleus
located in the concavity of macronucleus
•numerous food vacuoles
Movement:directional in rolling motion.
Cyst:Sub-spherical or oval cyst covered with a thick
cell wall,with Macronucleus and contractile in the
cytoplasm.

Pathology:
B. coli---- tissue invader ----hyaluronidase
Ulcer formed--- base is rounded and the neck is wide

*** Balantidiasis dysentery is similar to amoebic dysentery

Toxoplasma gondii

Stages of development:
•Tachyzoites- actively multiplying asexual form in
man;an obligate, intracellular parasite, pyriform
•Cyst-an accumulation of tachyzoites in a host cell
•male and female gametocytes.
•fertilized macrogametes develop into oocyst
•A mature cyst has two sporocyst.
Definitive host - cat where all stages of
development of the parasite are present

Mature oocyst - infective to other animals, man

*Trophozoites in the blood may also be transmitted

to the fetus by intra-uterine or congenital
transmission
*Toxoplasma crosses the placental barrier from the
mother’s blood
 Pathology:

T. gondii --- invade monocytes, reticulo-endothelial cells

and parenchyma or tissue cells--- destruction
•Most of the infections are asymptomatic
•In post-natally acquired infections, the clinical
manifestations include lymph adenopathy which maybe
febrile or afebrile, meningo-encephalitis and chorioretinitis
due to localization in the eyes
•In the intra-uterine or congenitally acquired infections
manifestations of the newborn include hydrocephalus,
epileptic seizures, chorioretinitis and cerebral calcifications
Diagnosis:
Specimen:Tissue biopsy materials---demonstrate
the organism
Serological tests maybe useful like indirect
hemeagglutination test for the circulating
antibodies, ELISA and Sabin-Feldman dye.
 Plasmodium vivax

Plasmodium falciparum

Plasmodium ovale

Plasmodium malariae
 Plasmodium
Four species of Plasmodium:
Plasmodium malariae (Laveran) 1881
Plasmodium vivax (Grassi and Feletti) 1890
Plasmodium falciparum (Welch) 1922
Plasmodium ovale (Stephens) 1922

Stages of development:
a. Trophozoite- earliest stage with one nucleus
b. Schizont- with nucleus divided into 8 – 24 nuclei each
nucleus enclosed by some cytoplasm forming
merozoites
c. Gametocytes Sexual forms with one large compact and
round or elongated nucleus.
Plasmodium falciparum
Malignant Tertian Malaria
Young trophozoite
Cytoplasm:Small, fine, pale blue ring
Chromatin:1 to 2 small red dots, some found in the
margin of RBC (Applique or Acole forms)
Size:1/5 to 1/3 diameter of RBC
Mature trophozoite
Cytoplasm:Thin, blue ring or comma-shaped
Chromatin:1 or 2 medium sized red dots
Schizont
Merozoites:8 – 24
Pigment:Dark brownish-black
Gametocytes
Shape:Banana/sickle/sausage/crescent
Color:Blue (male) or dense blue (female)
Nucleus:Reddish pink
Pigment:Few blue-black granules in the
center of the cytoplasm or scattered
RBC: Normal in size
Pigments: Maurer’s dots
Stages in peripheral blood: ring forms and gametocytes
Length of asexual cycle: 48 hours

Plasmodium vivax
Benign Tertian Malaria
Young trophozoite
Cytoplasm:Irregular blue, thick ring
Chromatin:Large red dots
Size:¼ to 2/3 of diameter of RBC
Mature trophozoite
Cytoplasm:Large, blue, irregular, small
particles of brownish orange pigment
Schizont
Merozoites:12 – 24
large, compact red granules
Gametocytes:
Female:Oval or rounded, dense blue with
dense red triangular nucleus; many
particles of orange pigment in cytoplasm
against the pale blue cytoplasm
Male:Rounded, pale blue with a round central pale
red nucleus; some particles of orange pigment in
cytoplasm
RBC: enlarged, pale
Pigments: Shuffner’s dots
Stages in peripheral blood: All
Length of asexual cycle: 48 hours
Plasmodium ovale
Benign tertian malaria
Young trophozoite
Cytoplasm:Regular, dense blue ring
Chromatin:Medium-sized red dot
Size:¼ to 2/3 of diameter of RBC
Mature trophozoite
Cytoplasm:Round, compact very blue with
a few particles of brown pigment
Chromatin:Large red dot
Schizont
Merozoites:8 – 14
large red granules in a rosette, round
and central mass of particles of
brown pigment
Gametocyte
Shape:Large, oval or round
Nucleus:1 round red spot
Pigment:Few brown particles in the
cytoplasm
RBC: enlarged often with torn/jagged end
or fringes or irregular edges
Pigments: James dots
Stages in peripheral blood : all
Length of asexual cycle: 48 hours

Plasmodium malariae
Quartan malaria
Young trophozoite
Cytoplasm:Thick, dense blue ring with some
granules of black pigments
Chromatin:1 large red dot
Size: ¼ to 2/3 of diameter of RBC
Mature trophozoite
Cytoplasm: either round, compact, dark blue with many
black particles of pigment
in band form
Chromatin:A round dot or a red band
Schizont
Merozoites:8 – 10 each one a large red spot
enclosed by pale cytoplasm
Gametocytes
Shape:Large, oval or rounded
Color:Female-Dense blue; male-pale blue
Nucleus:1 round spot of red chromatin
Pigment:
Large black granules in the cytoplasm
RBC : normal
Pigments : Ziemman’s dots
Stages in peripheral blood : all
Length of asexual cycle : 72 hours
Diagnosis:Specimen:Blood
a. Thick and thin blood smears stained with Giemsa or
Wright’s stain
b. Serological tests maybe used for diagnosis like IHA and
ELISA. The Quantitative Buffy Coat (QBC) method uses
a specially prepared capillary tube coated with acridine
orange. Malaria parasites take up this stain and appear
bright green and yellow when viewed under a
fluorescent microscope. The ParaSight F test is a
dipstick test for the simple and rapid diagnosis of P.
falciparum infection.
Thank you for
listening…….