PROTOZOA

Objectives
After taking this unit the student will be able to:

1. list common intestinal protozoa that affects human

2. Identify the common characteristics of intestinal protozoa

3. Explain the general characteristics of amoeba

4. Describe the basic structural and functional characteristics

of an ameba
 Outline

• Common feature of intestinal protozoa

• Introduction to Amoeba

• Important terminology

• Basic structural and functional characteristics of amoeba

• Taxonomic Classification of Amoeba

• locomotion and digestion of Amoeba

• respiration and reproduction of Amoeba

 INTESTINAL PROTOZOA
Ameba: Flagellates:
• Entamoeba histolytica • Giardia lamblia
• Entamoeba dispar • Dientamoeba fragilis
• Entamoeba coli • Chilomastix mesnili
• Entamoeba hartmanni • Trichomonas hominis
• Endolimax nana • Enteromonas hominis
• Iodamoeba bütschlii • Retortamonas intestinalis

Apicomplexa: Other:
• Cryptosporidium hominis • Blastocystis hominis
• Cryptosporidium parvum • Balantidium coli
• Cyclospora cayetanensis
• Isospora belli
 COMMON CHARACTERSTICS

TYPICAL FECAL-ORAL LIFE CYCLE

 Fecal-Oral Transmission
Factors
• Poor personal hygiene • Water-borne epidemics
• Food handlers • Male homosexuality
• Institutions • Oral-anal contact
• Children in day care • Zoonosis
centers • Entamoeba = no
• Developing countries • Cryptosporidium = yes
• Highly endemic • Giardia = controversial
• Poor sanitation
• Travelers diarrhea
 Control/Prevention

• Improve personal hygiene

•Protect water supply
• Especially institutions
•Treat water if
• Treat asymptomatic carriers
questionable
• Eg, family members
•Boiling
• Health education
•Iodine
• Hand-washing
•Not chlorin
• Sanitation
• Food handling
 Introduction

=Amoeba (plural amoebae)= Protists that moves by

means of pseudopods
=Amoeba = Greek word amoibe, means change
=Most Amoeba are free-living widely distributed mostly in
aquatic environment
fresh water - salt water,
wet soil, -on water plants leaves
=Few of the parasitic and free-living amebas produce
human disease
 General characteristics
• LOCOMOTION AND DIGESTION

– Locomotion and ingestion are accomplished by the use of the

pseudopodia.
– Once the food substance is contained within the cell, the
organism goes through the processes of enzymatically
breaking down and absorbing the nutrients.
– Egestion of unused residue is performed by the expulsion of
the vacuole out of the cell.
 RESPIRATION AND REPRODUCTION

• Respiration is performed by simple absorption of dissolved

oxygen from the liquid environment.
• Excretion of gases and waste is performed by diffusion out of the
organism through the cell membrane.
• Liquid regulation inside the body is controlled by Contractile
vacuoles
– serve as two-way pumps that control the hydrostasis between the
organism and the environment.

• These organisms reproduce through asexual reproduction

consisting of simple cell division.
 AMOEBIC STATE:
• Trophozoite

– Literally means any stage in the life cycle of a protozoan which

can ingest food.

• Cyst

– Is the non-motile form, which is protected by a distinct

membrane or a cyst wall.
– This is the infective stage of most Amoeba

• exception are Dientamoeba fragilis and Entamoeba

gingivalis
BASIC STRUCTURAL AND FUNCTIONAL
CHARACTERISTICS

Cell membrane
Oxygen and carbon dioxide enters and
leaves the ameba through cell membrane
IMPORTANT TERMS USED IN RELATION TO AMOEBAE
• Pseudopod
– Literally= false foot.
– temporary cytoplasmic processes formed at the surface of
the trophozoite
• Ectoplasm
– external hyaline portion of the cytoplasm which is
generally visible in a moving trophozoite
• Endoplasm
– Is the internal granular portion of the cytoplasm which
contains various food inclusions.
• Chromatoid body
– Is the RNA-protein complex which stains deeply with
basic dyes but not with iodine.
– It is found in the genus Entamoeba
• Glycogen vacuole
– Is a glycogen reserve which stains deeply with iodine
and is found mainly in the cysts.
• Food Vacuole
– A membrane bound vesicle in the cytoplasm formed
around an ingested food particle.
• Contractile vacuole
– A membrane bound vesicle in the cytoplasm which collects
and expels water from the cell. This is found in the free-
living amoebae.
• Chromatin
– Is the substances in the cell nucleus which stains with basic
dyes.

• Pre-Cyst
– Is the rounded form of trophozoite which precedes the
cystic stage. It differs from the cyst in not having a cyst
wall.
Excystation
– Is the process of emergence of the trophozoite from the
cyst.
Encystation
– Is the process of formation of the cyst from the
trophozoite.
Metacyst
– Is the trophozoite which emerges from the cyst..
 Taxonomy
• Phylum Sarcomastigophora
• Sub-Phylum Sarcodina
• Class Lobosea
– Order Amoebida
– Suborder Tubulina
• Genera: Entamoeba, Iodamoeba, Endolimax-
parasitic
• Suborder Acanthopodina
• Genera: Acanthamoeba - Free living
– Order Schizopyrenida
• Genera: Naegleria - Free living
Classification….
The Amoeba

Parasitic Ameba: Free living Ameba:

Intestinal Pathogenic
• Entamoeba histolytica • Naegleria fowleri
• Entamoeba dispar • Acanthamoeba spp
• Entamoeba coli • Balmuthia Mandrilarias
• Entamoeba hartmanni
• Endolimax nana
• Iodamoeba bütschlii
Mouth
• E. gingivalis
 Classification….
1- Pathogenic:
– intestinal amoeba: E- histolytica.
2- Non pathogenic:
►Mouth amoeba: E- gingivalis
►intestinal amoeba: E- coli, endolimax nana, iodamoeba
butschlii
 Summary
• Intestinal protozoa are all that inhabit the intestinaltruct
• Has atypical fecal-oral life cycle
• High prevalence in areas of poor sanitary condition
• Amoeba
– few are parasitic and causes diseases to humans
– Move by pseudopodiia
– exist both as trophozites and cyst
– Replliicatiion: biinary fiissiion
Entamoeba histolytica
After taking this lesson the student will be able to:

1. Discuses the epidemiology, life cycle, and clinical

aspects of E. histolytica

2. Discuses the pathology and pathogenic capabilities of

Entamoeba histolytica.
 Outline

E. histolytica
– History

– Epidemiology

– Morphology

– Transmission and life cycle

– Pathology and clinical manifestation

Entamoeba histolytica
History

1873.Alexandrewitch Lösch

describes amoebae associated

with severe dysentery in a patient

• He transferred amoebae to a dog by
rectal injection, which became ill and
showed ulceration of colon
• Patient who died from infection
showed similar ulcers upon autopsy
 Facultative Pathogenicity of E. histolytica

1925 Brumpt proposed two species: E. dysenteriae and E. dispar

1970's biochemical differences noted between invasive and non-
invasive isolates
80's/90's several antigenic and DNA differences demonstrated
• rRNA 2.2% sequence difference
1993 Diamond and Clark proposed a new species (E. dispar) to
describe non-invasive strains
1997 WHO accepted two species
 EPIDIMOLOGY

• Cosmopolitan distribution principally

• In tropical countries with warm climates and bad sanitary
conditions (fecalism).
• It is more frequent in poorest areas with contaminated water,
bad management of waste, and bad drainage system.
 Epidemiologic Risk Factors
Prevalence Severity
 lower socioeconomic status  children, esp. neonates
 crowding  pregnancy and
 lack of indoor plumbing postpartum states
 endemic area  corticosteroid use
 institutionalization  malignancy
 communal living  malnutrition
 promiscuity among male
homosexuals
Modified from Ravdin (1995) Clin. Inf. Dis. 20:1453
 FREQUENCY
• Amebiasis- is the 3rd leading cause of death worldwide
(malaria and schisto.)
~ 10% of world’s population may be
infected with E. histolytica / E. dispar.

• worldwide incidence = 0.2-50%(tropics)

• 500 million people worldwide infected
• 100 million people suffer acute symptoms
• 100,000 people die every year
• Note

• Since more survey to determine the prevalence are made on

stool examination which includes E. dispar
• Therefore, the current epidemiology of amoebiasis
doesn’t show the true prevalence of E histolytica
Molecular Epidemiology
• Molecular probes used to survey for E. Dispar and E.
Histolytica
• E. Dispar ~10-fold > E. Histolytica
• Discrete endemic pockets of E. Histolytica
• Many asymptomatic E.H. Infections
• ~10% of the E.H. Infections are associated with invasive
amebiasis
• ~25% seropositive for E. Histolytica in endemic areas
 Habitat and Morphology

• E. histolytica has both trophozoite and cyst forms.

• The trophozoites are microaerophilic,

– dwell in the lumen or wall of the colon,

– feed on bacteria and tissue cells,

– and multiply rapidly in the anaerobic environment of the gut.

– When diarrhea occurs, the trophozoites are passed unchanged

in the liquid stool.
Electron microscopic demonstrate
microfilaments,
an external glycocalyx, and
cytoplasmic projections thought
to be important for attachment
Cysts of Entamoeba histolytica/E. dispar,
 Cont..
• Pseudopodia are fi nger-like projections
formed by sudden jerky movements of
ectoplasm in one direction
• The direction of movement may be changed
• Nucleus is spherical 4–6 µm in size and
contains central karoyosome
• The nuclear membrane is lined by a rim of
chromatin distributed evenly as small granules
 Cystic Stage
• Spherical in shape about 10–20 µm in size.
• The early cyst contains a single nucleus and
two other structures—a mass of glycogen and
1–4 chromatoid bodies or chromidial bars,
The chromatoid bodies are so called because
they stain with hematoxylin, like chromatin.
• The mature cyst is, thus quadrinucleate
 Cont..
• Erythrocytes ingestion is diagnostic as
phagocytosed red cells are not found in any
other commensal
• The trophozoites divide by binary fi ssion in
every 8 hours.
• Trophozoites survive upto 5 hours at 37°C and
are killed by drying, heat, and chemical
sterilization.
 E. Histolytica Life Cycle
Definitive Host: Humans
Intermediate Host: None
• Transmission
• Ingestion of mature cyst ( 10days)
• The average infective dose exceeds 1000 organisms, but
• Ingestion of a single cyst has been known to produce
infection.
• Location in host:
• Colonization is most intense in areas of fecal stasis such as
the cecum and rectosigmoid but may be found throughout the
large bowel.
• May be carried to liver, lungs, and other body parts if it
perforates the intestines
 Cont…
• As the cyst wall is resistant to action of gastric
juice, the cysts pass through the stomach
undamaged
• Excystation: When the cyst reaches caecum
or lower part of the ileum, due to the alkaline
medium, the cyst wall is damaged by trypsin
Life cycle….

Humans are the only reservoir excreting up to 45

million cysts daily

Mature cysts can survive in envt.

•temp up to 55°C,
•Norm Chlorine conctentration
•gastric acid.
 Excystation

• In response to gastric acid, ingested

cysts release trophozoites
• in the upper intestine occurs in small
intestine
• nuclear division (48)
• cytoplasmic divisions (8 amebala)
• trophozoites migrate to large
intestine
• Trophozoites colonize
large intestine
• Feed on bacteria and
debris
• Replicate by binary fission
 Encystation
• Trophozoite rounds up
• secretion of cyst wall
• aggregation of ribosomes (=
chromatoid bodies)
• 2 rounds of nuclear division
(14 nuclei)
• survive weeks to months
 PATHOGENESIS and PATHOLOGY OF
AMEBIASIS
• NON-INVASIVE
– ameba colony on intestinal mucosa
– asymptomatic cyst passer
– non-dysenteric diarrhea, abdominal cramps, other
GI symptoms
• INVASIVE
– necrosis of mucosa  ulcers, dysentery
– ulcer enlargement  severe dysentery, colitis,
peritonitis
– Metastasis  extra-intestinal amebiasis
 NON-INVASIVE
1. Trophozoites
• colonize , adhere to the mucus layer and ingest bacteria and
cellular debris from the lumen.
• Usually asymptomatic, or
• Exhibits symptoms ranging from mild abdominal discomfort
to diarrhea and cramps..
INVASIVE
• The non-invasive infection can persist or progress to an
invasive by
• Penetration of mucus layer
• Contact-dependent killing of epithelial cells
• Breakdown of tissues (extracellular matrix)
• Contact-dependent killing of neutrophils, leukocytes, etc.
• Three key virulence factors:
– Amebic lectin: binds parasite to galactose-containing

sugars on host cells

– Amoebapores: adherence-dependent cytolysis

– Cysteine protease: cleaves antibodies and C3

– Trophozoites ingest human cells

• ulcers with raised borders
• Mucosa between ulcers appears normal
• little inflammation between lesions
Once the lesion penetrates below the superficial epithelium, it
meets the resistance of the colonic musculature and spreads
laterally in thesubmucosa

• ‘flasked-shaped ulcer’ trophozoites at boundary of

necrotic and healthy tissue
• trophozoites ingesting host cells
• dysentery (blood and mucus in feces)
‘hematophagous’
trophozoites
Ulcer Enlargement can lead to secondary
infection and extraintestinal lesions

• Ameba expand laterally and downward

into lamina propria
• Localized sloughing (ulcers coalesce)
• Perforation of intestinal wall
• Peritonitis
• 2o bacterial infections
• Local abscesses
• Ameboma (=amebic granuloma)
ameboma = inflammatory thickening
of intestinal wall around the abscess
(can be confused with tumor)
 Lateral and Downward Expansion
of Ameba into Lamina Propria
• localized sloughing • ulcers coalesce

• perforation of intestinal wall

Gross pathology of large intestine
due to Entameba histolytica
Extraintestinal amebiasis
• Metastasis via blood stream
• Primarily liver (portal vein)
• Other sites less frequent
• Ameba-free stools common
• High antibody titers
Amebic liver abscess
• Most common form of
extraintestinal amebiasis
• symptoms are right upper
quadrant pain and fever
• Acute as well as chronic illness,
with gradual or sudden onset
Amebic Liver Abscess
• Fast growing abscess filled
with:
• chocolate-colored ‘pus’
• necrotic material
• usually bacteria free
• amoebae are found only
at borders
• lesions expand and
coalesce
• further metastasis, direct
extension or fistula
Amebic liver abscess
• 30-50% of patients with liver abscess
show also pneumonic involvement
• Rupture is again a major thread,
especially rupture into the
pericardium
• Draining abscesses is today only
performed in extreme cases when
rupture is feared
• Responds well to chemotherap y
Pulmonary Amebiasis
• rarely primary
• rupture of liver abscess
through diaphragm
• 2o bacterial infections
common
• fever, cough, dyspnea,
pain, vomica
Cutaneous Amebiasis
• intestinal or hepatic fistula
• mucosa bathed in fluids
containing trophozoites
• perianal ulcers
• urogenital (eg, labia,
vagina, penis)
 CLINICAL MANIFESTATION
• Amebiasis presents a wide range of clinical syndromes
• Depends on:
– host’s previous exposure to parasite
• Chronic, low-level exposure- asymptomatic.
• Less frequent exposure- severe symptoms.
– on nutritional status of host
– what organ the parasite invades.
• Abdominal cavity
– Peritonitis, abdominal pain, cramping, and anemia
• Liver
– Symptoms are similar to hepatitis
• Lungs, brains, or heart
– May cause the death of the host.
• The incubation period
– Range from a few days to months or years with 2-4
weeks being the most common.
• Transitions from one type of intestinal syndrome to
another can occur and
• Intestinal infections can give rise to extraintestinal
infections.
 Clinical Syndromes Intestinal Symptoms
Associated with Amebiasis range
Intestinal Disease • mild to intense
 asymptomatic cyst passer • transient to long lasting
 symptomatic nondysenteric • Non dysenteric
infection • Diarrhea, - cramps,
 amebic dysentery • Flatulence, - nausea
 fulminant colitis • dysenteric
 + perforation (peritonitis) • blood/mucus in stools
 ameboma (amebic granuloma) • cramps/pain, tenesmus
 perianal ulceration • ameboma
• palpable mass, obstruction
Extraintestinal Disease Liver symptoms
 liver abscess Symptoms are similar to hepatitis
 pleuropulmonary amebiasis Lungs, brains, or heart
 brain and other organs May cause the death of the host
 cutaneous and genital diseases
Metronidazole is the drug of choice for extra-
intestinal amebiasis
• Several drugs are available to clear
symptomatic and asymptomatic enteric
(luminal) infection
• Metronidazole (Flagyl) is the drug of
choice for invasive amoebiasis (and
should be combined with a lumen acting
drug as it is not fully effective on luminal
stages)
• Metronidazole is a prodrug which is
activated by an enzyme involved in the
microaerobic fermentation metabolism of
E. histolytica
 Summary
E. histolytica
• Is the most medically relevant amoeba
species that causes greatest human disease
• Has world wide distribution with high
frequency in countries with poor sanitary
conditions.
• Recently separated from the
morphologicaly identical but non pathogenic
E.dispar
– Causes Amebiasis most with mild or no symptoms
but can cause a serious invasive disease:
 Summary
E. histolytica
• Transmission is through ingestion of cyst stage in
contaminated food/ water.
• The Lumen of colon and cecum are the usual
habitat but can also invade extra-intestinal
locations including liver, lung, brain, skin, and
other tissues.
• Trophozoites or cysts is the diagnostic stage.
Non pathogenic Amebae
in Human Intestines

Entamoeba hartmanni
Entamoeba dispar
Entamoeba coli
Endolimax nana
Iodamoeba bütschlii
 Learning Objectives
• Upon completion of this unit of instruction and
lecture, the student will be able to:
– Discuss epidemiology and morphology of non
pathogenic amoeba
– llustrate the life cycle and clinical aspects of the
non pathogenic amoeba
 Non pathogenic Amebae in Human Intestines

Outline
– Geographical distribution
– Morphology
– Transmission and life cycle
– clinical manifestation
Entamoeba • Geographic distribution:
hartmanni Cosmopolitan
• Definitive Host: Humans
• Intermediate Host: None
• Mode of transmission:
Ingestion of food and water
contaminated by feces.
 E. hartmanni Life Cycle
Trophs in
large
intestines

Binary
Ingested Fission Cyst

Out into
environment
Change in chemistry as troph moves down colon
tells the troph to form a cyst.
• Location in definitive host: Cecum and large
intestine
• Pathology and Treatment: None.
– Harmless commensal that does not lyse host
cells.
– Eats bacteria in intestines.
• Diagnosis: Important to distinguish between it
and E.histolytica
– E. hartmanni is much smaller
 Entamoeba hartmanni
• cysts
• 6-8 m
• 4 nuclei (mature)
• blunt chromatoid bodies
• CB persist in mature cysts
• trophozoites
• 8-10 m
• nuclear structure
• peripheral chromatin
• small karyosome
• < 10 m = E. hartmanni
• > 10 m = E. histolytica
• ‘small race’ of E. histolytica
• designated species in 1957
 Entamoeba coli
• Definitive Host: Humans
• Intermediate Host: None
• Geographic distribution: Cosmopolitan
– Much more common than E. histolytica
• Mode of transmission: Ingestion of contaminated
food and water.
 E. coli Life Cycle
Trophs in
large
intestines

Binary
Ingested Fission Cyst

Out into
environment
Change in chemistry as troph moves down colon
tells the troph to form a cyst.
• Location in Definitive host: Cecum and large
intestine.
• Pathology and Treatment: None
– Harmless commensal
– Feeds on bacteria, yeast, and wastes in
intestines
• Diagnosis: Important to distinguish between it
and E. histolytica
– Avoid unnecessary treatment
– 8 nuclei in cyst vs. 4 in E. histolytica
– Different shape nuclei.
Entamoeba coli
• trophozoites
• 20-25 m
• broad blunt pseudopodia
• nuclear structure
• peripheral chromatin
• small karyosome
• irregular peripheral chromatin?
• eccentric karyosome?
Entamoeba coli
• cysts
• 15-25 m
• 8 nuclei (mature)
• pointed chromatoid bodies (less
prominent)
Entamoeba
coli - Cyst
• Usually more than 4, up
to 8 nuclei.
• Chromatin bars (C) have
splinter-like ends (below)
C
 Endolimax nana

• Definitive Host: Humans

• Intermediate Host: None
• Geographic distribution: Cosmopolitan
• Mode of transmission: Ingestion of contaminated
food and water.
 E. nana Life Cycle
Trophs in
large
intestines

Binary
Ingested Cyst
Fission

Out into
environment
Change in chemistry as troph moves down colon
tells the troph to form a cyst.
• Location in Definitive host: Cecum and large
intestine.
• Pathology and Treatment: None
– Harmless commensal
– Feeds on bacteria
• Diagnosis: Important to distinguish between it
and E. histolytica
– Avoid unnecessary treatment
– Much smaller with different nuclei
Endolimax nana
• cysts
• 6-8 m
• 4 nuclei
• trophozoites
• 8-10 m
• nuclear structure
• no peripheral chromatin
• large karyosome
 Endolimax nana -
troph
• Large endosome in the nucleus
• Very small size
 Endolimax
nana - cyst
• Very small size.
• Up to four nuclei
– With very large endosome.
 Iodamoeba buetschlii
• Definitive Host: Humans, other primates, and pigs.
– Most common in pigs
• Probably the original host
• Intermediate Host: None
• Geographic distribution: Cosmopolitan
• Mode of transmission: Ingestion of contaminated
food and water.
 I. buetschlii Life Cycle
Trophs in
large
intestines

Binary
Ingested Cyst
Fission

Out into
environment
Change in chemistry as troph moves down colon
tells the troph to form a cyst.
• Location in Definitive host: Cecum and large
intestine.
• Pathology and Treatment: None
– Harmless commensal
– Feeds on bacteria, yeast, and wastes in
intestines
• Diagnosis: Important to distinguish between it
and E. histolytica
– Avoid unnecessary treatment
– Large, clear vacoule in trophs and cysts
– Different shape nuclei.
Iodamoeba
bütschlii
• cysts
• 10-12 m
• 1 nucleus
• glycogen vacuole
• trophozoites
• 12-15 m
• nuclear structure
• no peripheral chromatin
• large karyosome
 Iodamoeba
butschlii - troph
• Larger than E. nana
• Large endosome in
nucleus
• Many clear vacoules
 Iodamoeba
butschlii - cyst
• Very similar to E. nana but
larger
• Large endosome in single
nucleus
• Large, single, clear vacoule.
 Entamoeba gingivalis
• Definitive Host: Humans. Also other primates, dogs,
and cats.
• Intermediate Host: None
• Geographic distribution: Cosmopolitan
– About 50% of the population have it.
– Up to 95% of “unhealthy” mouths have it.
• Mode of transmission: Direct oral contact (kissing).
 E. gingivalis Life Cycle
Trophs in
mouth

Binary Direct oral

Fission contact passes
it to next host.
No cyst stage. Never goes
out to external environment.
• Location in Definitive host: Mouth,
particularly along gums line. Also in tonsils.
• Pathology and Treatment: None
– More common in mouths with gingivitis and
tonsillitis
– Probably doesn’t cause disease
• Most likely due to abundance of food in “dirty”
mouth.
• Diagnosis: Sample of scrapings from teeth
Other Entamoeba
E. gingivalis
• oral cavity
• no cyst stage
• trophozoites nearly identical to E.
histolytica
• periodontal disease?
 Remember
1. Definitive Host
2. Intermediate host
3. Geographic distribution
4. Mode of transmission
5. Location in definitive host
6. Pathology
• Disease, symptoms, diagnosis,
treatment
7. Life Cycle
 Laboratory Learning Objectives:
• Upon completion of this unit of instruction and laboratory
experience, the student will be able to:
– Differentiate and identify Amoebas by stage, genus, and
species
– Differentiate cyst forms from trophozoite forms of E.
histolytica
– Distinguish trophozoite and cyst forms of E. histolytica from
other non pathogenic amoeba and artifacts in stool spesmen
– Identify the specimen of choice for recovery of specific protozoan
organisms.
– Identify the special technique required for recovery of specific protozoan
organisms.
– Describe the Microscopy, Immunodiagnosis, Molecular and Culture
based diagnosis of Amoeba
 Outline
• Basic guidelines
• Microscopic examination of faecal specimens
• Differential characteristics of amoeba by: -
– stage(trophozoites/cyst)
– Genus
– species
• Immunodiagnosis
– Antigen detection
– Antibody detection
• Molecular-based diagnosis
• Culture method
 Basic guidelines
A. Multiple stool samples (at least 3) should be tested before
a negative result is reported

A. To maximize recovery of cysts, stool samples in formalin,

or other fixatives, should be concentrated prior to
microscopic examination (e.g.,10 min at 500 × g when
using the formalin-ethylacetate concentration procedure)
Basic guidelines
C. Exception: Specimens to be used for EIA or
rapid cartridge assays should NOT be
concentrated because antigens are lost during the
procedure!
D. Choice of diagnostic techniques depends on
available equipment and reagents, experience, and
considerations of time and cost.
Microscopy
Intestinal amoebiasis
1. Microscopic examination
A. Examination of fresh dysenteric faecal specimen
or rectal scraping for motile trophozoites
• wet mounts
– Fresh stool
• Permanently stained preparations (e.g.
trichrome).
– Fresh stool
– Preserved stool
Microscopic examination

B. Examination of formed or semi-formed faecal

specimen for cysts
– Fresh stool
– Smear after concentration
– Preserved stool
 Microscopic examination

• Correct identification Amoeba depends on:

Proper collection of stool and preparation

of slides
Systematic microscope technique
Knowing and observing the morphology of
the species.
Specimen Collection and processing
• Collect the stool in a dry, clean, leak proof container.
• Make sure no urine, water, soil or other material gets
in the container.
• Fresh stool should be examined, processed, or
preserved immediately.
• Preserve the specimen as soon as possible in two
different preservatives(10% formalin and PVA
(polyvinyl-alcohol …),
• Specimen collection may need to be repeated if the
first examination is negative.
– If possible, three specimens passed at intervals of
2-3 days should be examined
 COLLECTION OF STOOL
TROPHOZOITE
CYST

5% OR 10%
FORMALIN PVA/SAF

PERMENANT
WET MOUNT STAINED SMEAR
CONCENTRATION
Trichrome or
Iron haematoxylin
TROPHOZOITE
OR CYST

GENERAL PRECISE
MORPHOLOGY MORPHOLOGY
ribution of protozoa in relation to stool consist
 PREPARATION OF SLIDES

• In many instances amebae can be identified by

examining two types of preparations;

– 1. Initial Wet Mount Slide

– 2. Concentrate Wet Mount Slide
2. Permanent Stain Slide, IF:
A. The presence of trophozoites is suggested by
the consistency (watery, loose, soft) of fresh
specimen and the presence of blood and/or
mucus
B. Necessary for the identification of organisms
unidentified in the wet mounts
C. A permanent record is desired
3. Culture Slides (wet mount and permanent
stain):
– IF culture has been prepared as a result of earlier
indication of possible presence of amebae
TEMPORARY WET MOUNTS
• Three general types of solutions (reagents)
– saline sustains both trophozoites and cysts
– Dobell's iodine makes clearer nuclei in cysts

• The Initial (Formalin), Concentrate, and Culture

Wet Mounts do not require saline since the
samples for them are liquid enough.
 PREPARATION OF PERMANENT STAIN SLIDES

The slide is prepared:

A. If consistency of the fresh specimen and the
presence of blood and/or mucus suggest the
presence of trophozoites,
B. When necessary for identifying organisms
unidentified in the wet mounts, and
C. For permanent record
PERMANENT STAIN SLIDES CAN BE PREPARED
Systematic microscope technique
–1
 Most Amoeba exist as:
STAGE (trophozoite or cyst) is determined by the overall shape of the
organism and the presence of certain bodies in its cytoplasm.

-contain bacteria and food particles - -chromatoid bodies

contain RBC -glycogen vacuole
-show motility -inclusion mass M
Most have -smooth and rounded
-more irregular shape -more than one nucleus
-uninuclear
GENUS is determined by the presence or absence of PERIPHERAL
CHROMATIN in the nucleus and/or CHROMATOlD BODIES in the cytoplasm.
Characteristics used to
distinguish species
Nuclear Morphology(trophozoite & Cyst)
Characteristics used to distinguish species of
intestinal amebae
Trophozoites
• Motility- progressive or nonprogressive.
• Cytoplasm
– Appearance-finely granular, coarsely
granular, or vacuolated.
– Inclusions-erythrocytes, bacteria,
molds.
• Nucleus
– Number present.
– Peripheral chromatin- present or absent. If present,
the distribution along the inner surface of the
nuclear membrane and the size of the granules are
important.
– Karyosome- location and size.
• Size
– Sizes overlap, but they can be used as a secondary
distinguishing feature.
– Size is the chief criterion for distinguishing
Entamoeba histolytica from Entamoeba hartmanni.
• Cysts
– Nucleus
• Number present.
• Peripheral chromatin-present or absent.
• If present, the distribution of the granules along the
inner surface of the nuclear membrane
• Karyosome- location and size.
– Cytoplasm
• Chromatoid bodies-present or absent.
• If present, the shape is important.
• Glycogen- appearance.
• Size
– Sizes overlap, but they can be used as a
secondary distinguishing feature.
– Size is the major criterion for distinguishing
Entamoeba histolytica from Entamoeba
hartmanni.
• Shape
– Shapes vary, but they may be useful as a
secondary distinguishing feature.
 5. Identify Species
Type of motility
Type of Pseudopodia
ve
s ti
ge
g
Su

y
or
at
rm
nfi
Co
 Characteristics used to distinguish
species
Cysts
Size

Shape
No of Nucleus
• Both unstained and stained preparations are
necessary to demonstrate all of the features.
• Species identification of trophozoites can rarely
be made from single features, such as nucleus or
cytoplasm, or from a single organism.
• Several features and several organisms should be
examined.
• Cysts are less variable and can usually be more
easily identified than trophozoites.
Trichrome stain
• Trophozoites
– measure 15 to 20 μm (range may be 10 to 60 μm).
– Presence of one nucleus with evenly arranged chromatin on the
nuclear membrane and
– a small, centrally located karyosome are morphological features
of trophozoites.
– The cytoplasm is finely granular and few ingested bacteria or
debris may be present.
– Presence of red blood cells within the cytoplasm of trophozoites
is a diagnostic feature for the identification of E. histolytica.
– Ingested RBCs are not frequently seen; in the absence of this
diagnostic characteristic E. histolytica/E. dispar should be
reported.
E.histolytica
• Trophozoite
– 15 to 20 µm (range 10 to 60
µm
– has a single nucleus,
– centrally placed karyosome
and
– uniformly distributed
peripheral chromatin.
– has a cytoplasm with
granular or "ground- glass"
appearance
Entamoeba histolytica/E. dispar,

• Cysts
– Measure 12 to 15 µm.
– are usually spherical
– Mature cysts have 4 nuclei.
– The nuclei have
characteristically centrally
located karyosomes,
– And fine, uniformly
distributed peripheral
chromatin.
– chromatoid bodies with
typically blunted ends.
Trichrome stain
• Cysts
– 12 to 15 μm (range 10 to 20 μm) and
– have 1 to 4 nuclei. Chromatoid bodies
– with bluntly rounded ends may also be present.
Entamoeba cysts (light microscopy)

E. coli E. histolytica
 SIMILAR ORGANISMS AND ARTIFACTS

• Some of the most difficult problems in the correct

identification of amebae in stool specimens are caused by:
– the presence of free-Iiving amebae, other protozoa, and
the many confusing elements whose structures are similar
to those of amebae.
– Some of these "artifacts" are pictured below.
– Look closely and note how their characteristics differ from
the classic amebae you have learned:
 Low sensitivity of microscopic
examination
• Many E.histolytica infections were confused with
E.dispar
• The only way to distinguish E.dispar from E.histolytica
microscopically is erythrophagocytosis.
• False-positive results due to misidentification of
macrophages and nonpathogenic species of
Entamoeba
 Immunodiagnosis
1. Antibody Detection
• Amoebiasis can be diagnosed by a serological test.
– Because anti-E. histolytica antibodies are produced by
hosts infected with E. histolytica but not by carriers of E.
dispar,
– Of patients with current cases of amoebiasis, 75% - 85%
have IgG anti-E. histolytica antibodies present in their
blood.
– This technique is used most often for diagnosing chronic
or extra-intestinal infections
• TECHNIQUE
– IHA test, Enzyme immunoassay (EIA) etc..
– IHA test has been replaced by EIA test kits for
routine serodiagnosis .
– The EIA test detects antibody specific for E.
histolytica in :
• 95% of patients with extraintestinal amebiasis,
• 70% of patients with active intestinal infection, and
• 10% of asymptomatic persons who are passing
cysts of E. histolytica
• The persistence of the antibodies
– Detectable IgG antibody levels can remain high for
years after the infection, and
– In 90% of people who have recovered from a case
of amoebaisis.
– the serological test can only be used to determine
if a person has ever had amoebiasis and
– gives no indication of the current state of the
infection. in areas where amoebiasis is endemic
• Currently, research is being done on an anti-E.
histolytica IgM antibody that is present in patients
with chronic infections, but does not persist in the
serum after recovery
2. Antigen Detection
– Useful to distinguish between pathogenic and
nonpathogenic infections.
– Using monoclonal antibodies, it can distinguish
between E. histolytica and E. dispar infections.
– Currently kit is available:
• which detects only pathogenic E. histolytica infection
in stool;
• which detect E. histolytica antigens in stool but do
not exclude E. dispar infections.
– Antigen detection using ELISA can be performed
rapidly and easily, making it the diagnostic test
method of choice for clinical use in the
developing world
 Enzyme immunoassays (EIA)
• Immunoassay kits are commercially available that detect E.
histolytica.
• Currently, these tests require the use of fresh or frozen stool
specimens and cannot be used with preserved specimens.
 Rapid immunochromatographic cartridge
assay
• A rapid cartridge is available that detects antigens of
E. histolytica/E. dispar,
– however this assay does not distinguish between E.
histolytica and E. dispar.
– This assay also detects antigens of Giardia and
Cryptosporidium.
• Stool samples must be fresh or frozen and should not
be concentrated prior to testing.
• Borderline positives and questionable negatives
should be further confirmed by additional testing.
• This assay is quick and easy to perform and no
special equipment is needed.
 Cultivation of Amoeba
• LABORATORY GROWTH
– Trophozoites are facultative anaerobes that
require complex media for growth.
– Most require the addition of live bacteria for
successful isolation.
– Sterile culture techniques (axenic) have been
developed, however, and
– are essential for the preparation of the purified
antigens required for serologic testing,
zymodeme typing, and characterization of
virulence factors.
– Such techniques are generally available only in
research laboratories.
 Cultivation of Amoeba
• Cultivation increases the possibilities of finding an
organism by permitting the amebae to multiply.
• Cultures are primarily research tools rather than
diagnostic tools.
– Culture method is not currently available and not
readily usable for routine diagnosis
– It requires a week to complete and are negative in
many microscopy-positive samples,
– in some cases due to delays in sample processing or
due to the institution of antiamebic therapy prior to
stool collection
 Cultivation of Amoeba

Xenic in which the parasite is grown in the presence of an

undefined flora. D. fragilis ,B. coli, E. histolytica and B. hominis

Monoxenic in which the parasite is grown in the presence of a

single additional species E. histolytica and B. coli
Axenic in which the parasite is grown in the absence of any
other metabolizing cells. G. intestinalis, E.histolytica and
B.hominis
Isoenzyme analysis of cultured Amebae enables the
differentiation of E. histolytica from E. dispar
 ENTAMOEBA HISTOLYTICA
And other intestinal amoebae
 Molecular technique
• Polymerase Chain Reaction (PCR) can be used to
amplify specific strands of E. histolytica DNA or RNA,
so that it can be more easily observed in dilute
samples (especially environmental samples.)
• PCR methods offer sensitivity and specificity
• It may be not well suited for use in developing
countries where amoebiais is endemic as it is time-
consuming, cumbersome, and expensive
• Moreover.
– E.histolytica trophozoites can be isolated from tissue
biopsies or ulcer scrapings.
– Barium enemas can be used to view abscesses, ulcers, and
amoebomas in the colon, and ultrasound, CT and MRI
scans of the abdomen can help identify liver abcesses.
Extraintestinal (hepatic)
 symptoms
history of dysentery
RUQ pain
enlarged liver
 serology (current or past?)
 imaging (CT, MRI, ultrasound)
 abscess aspiration
only select cases
reddish brown liquid
 trophozoites at abscess wall
Sigmoidoscopic examinations.

• When amebiasis is suspected and stool

samples are negative, material for
examination may be collected by
sigmoidoscopy.
•The procedure should be performed after a
normal bowel movement or two to three
hours after administration of a cathartic.
•The specimen is collected with a pipette
rather than with a swab by aspirating
material from any visible lesion and from
the mucosa.
•Examination of sigmoidoscopic material
should be done immediately, but fixation
Liver abscesses.

•Exudates aspirated from the liver may

contain trophozoites of Entamoeba
histolytica.
•The first portion of the specimen does
not usually yield organisms, but the
second (reddish) portion does.
•Aspirates should be examined
microscopically with wet preparations
as well as with permanent stains.
 Non pathogenic
Amebae in Human
Intestines
Entamoeba coli
Entamoeba hartmanni
Endolimax nana
Iodamoeba bütschlii
 Entamoeba hartmanni
• cysts
• 6-8 m
• 4 nuclei (mature)
• blunt chromatoid bodies
• CB persist in mature cysts
• trophozoites
• 8-10 m
• nuclear structure
• peripheral chromatin
• small karyosome
• < 10 m = E. hartmanni
• > 10 m = E. histolytica
• ‘small race’ of E. histolytica
• designated species in 1957
Entamoeba coli
• trophozoites
• 20-25 m
• broad blunt pseudopodia
• nuclear structure
• peripheral chromatin
• small karyosome
• irregular peripheral chromatin?
• eccentric karyosome?
Entamoeba coli
• cysts
• 15-25 m
• 8 nuclei (mature)
• pointed chromatoid bodies (less
prominent)
Entamoeba
coli - Cyst
• Usually more than 4, up
to 8 nuclei.
• Chromatin bars (C) have
splinter-like ends (below)
C
Endolimax nana
• cysts
• 6-8 m
• 4 nuclei
• trophozoites
• 8-10 m
• nuclear structure
• no peripheral chromatin
• large karyosome
 Endolimax nana -
troph
• Large endosome in the nucleus
• Very small size
 Endolimax
nana - cyst
• Very small size.
• Up to four nuclei
– With very large endosome.
Iodamoeba
bütschlii
• cysts
• 10-12 m
• 1 nucleus
• glycogen vacuole
• trophozoites
• 12-15 m
• nuclear structure
• no peripheral chromatin
• large karyosome
 Iodamoeba
butschlii - troph
• Larger than E. nana
• Large endosome in
nucleus
• Many clear vacoules
 Iodamoeba
butschlii - cyst
• Very similar to E. nana but
larger
• Large endosome in single
nucleus
• Large, single, clear vacoule.
Other Entamoeba
E. gingivalis
• oral cavity
• no cyst stage
• trophozoites nearly identical to E.
histolytica
• periodontal disease?
 Table 3.1: Differential characteristics of intestinal amoebae (live specimens)

E.
D.fragilis
- histolyt E. hartmanni E. coli E. polecki E.nana l. butschlii
*
ica

Active
Active
especial
often
ly in
TraphozoiteMovement Sluggish Sluggish sluggish Sluggish Active multiple
acute
pseudopo
dysente
ds
ry

Red
cell in
case of No red
Inclusions No red cells No red cells No red cell No red cell No red cell
tissue cell
invasio
n
 E.
- E. histolytica E. coli E. polecki E. nana I.butschlii D.fragilis
hartmanni

TrophozoiteNo. of
1 1 1 1 1 1 2
nuclei

Large
Usually Usually Large
Karysome(Nucleol Usually Usually may be
central central Large generally
us) central small central small fragment
small small irregular
ed

Usually
Usually Usually Usually Usually
Peripheral symmet Incon- Incon-
symmetrical symmetri symmetrical symmetrical
chromatin rical spicuous spicuous
fine cal fine fine fine
fine