An overview of general morphology of Protozoa and detailed content on Entamoeba histolytica

1. PROTOZOA –OVERVIEW
By
Dr. J. Neena Priya
Assistant Professor, Dept of Microbiology,
Dr. MGR JANAKI COLLEGE

2. • Protozoa - from the Greek “protos” and “zoon” meaning “first animal”,
are members of eukaryotic protists
• Protozoa are found in all moist habitats. They are common in sea, in soil
and in fresh water.
• These organisms occur generally as a single cell.
• A resistant cyst (non-motile) stage to survive adverse environmental
conditions, such as desiccation, low nutrient supply, and even
anaerobiosis.
Morphology
• ranging in size from 2 -100μm
• within a mass of protoplasm, consisting of a true membrane – bound
nucleus and cytoplasm.
• nucleus contains clumped or dispersed chromatin

3. • Protozoa are ubiquitous in moist areas, including the human alimentary
canal.
• Protozoa may be divided into free-living forms and symbiotic forms
• Entamoeba histolytica may invade tissue and produce disease.
• The majority of ciliates are free living and seldom parasitize humans.
• Flagellates of the genus Trypanosomes and Leishmania are capable of
invading the blood & tissue of humans, where they produce severe
chronic illness.
• Others such as Trichomonas vaginalis and Giardia lamblia, inhabit
urogenital and gastrointestinal tracts and initiate disease.
• Sporozoan organisms, in contrast, produce two of the most potentially
lethal diseases of humankind: Malaria and Toxoplasmosis

4. Reproduction
• protozoa multiply by asexual reproduction
• Some parasitic forms may have an asexual phase in one host and a sexual phase in
another host
• Pathogenic protozoa can spread from one infected person to another by:
• Faecal – oral transmission of contaminated foods and water.
• Insect bit inoculums or rubbing infected insect faeces on the site of bite
Pathogenesis
Factors that are important for pathogenicity include:
• Attachment to the host
↓
replication to establish colonization.
↓
• Toxic products released by parasitic protozoa.
↓
• Shifting of antigenic expression to evade the immune response and inactivate host
defences

5. • Protozoa of medical importance are classified based on their morphology
and locomotive system as described below:
• Amoebas - Entamoeba histolytica
• Flagellates - Giardia lamblia, Trichomonas vaginalis, Trypanosoma spp,
Leishmania spp
• Ciliophora - Balantidium coli
• Coccidian - Isospora belli, Cryptosporidium parvum, Toxoplasma gondii,
Plasmodium species
• Protozoan pathogens can also be grouped according to the location in
the body where they most frequently cause disease

6. Entamoeba histolytica
AMOEBIASIS

7. Entamoeba histolytica - AMOEBIASIS
• Entamoeba histolytica is the causative agent of Amoebic dysentery or
Amoebiasis infects more than 10% World’s population.
• Dysentery affects the intestines that results in intense diarrhoea with blood
and is often accompanied by pain and fever.
• Amoebiasis is endemic in India and affects all age groups.
Morphology of Entamoeba histolytica
• There are two stages in the life cycle :
1. actively growing and feeding stage referred to as the Trophozoite form
2. transmission stage called the Cyst form
• Trophozoite – actively motile feeding stage.
• Cyst – resistant, infective stage.

8. (a) Trophozoites
• vary in size from about 10-60μm in diameter.
• The cell body is divisible into two distinct portions—Ectoplasm and Endoplasm.
• The ectoplasm is clear and translucent while the endoplasm is granular.
• The endoplasm often contains ingested red blood corpuscles.
• The pseudopodia may be long, finger like or rounded in shape.
• The nucleus is indistinct in living condition but in stained
preparation it shows a central Karyosome or Endosome
• A uniform ring of peripheral chromatid granules.
• The nuclear membrane is very delicate.
• The nucleus is 4-6μm in diameter.
• In the case of dysentery, however, RBCs may be
visible in the cytoplasm
• This feature is diagnostic for E. histolytica.

9. (b) Cyst
• Cysts range in size from 10-20μm. The cysts are spherical. The cyst wall is
double and the cytoplasm usually bears four nuclei.
• The immature cyst has inclusions namely; glycogen mass and chromatid bars.
• As the cyst matures, the glycogen completely disappears.

10. Infective form:
• Mature quadrinucleate cyst; it is spherical in shape with refractile wall
Quadrinucleate cyst of Entamoeba histolytica
Habitat:
• Trophozoites of E. histolytica live in the mucosal and submucosal layers of
the large intestine of man.
• Life cycle of Entamoeba histolytica has two-stage:
• motile trophozoite and non-motile cyst.
• Trophozoites are found in intestinal lesions, extra-intestinal lesions, and
diarrheal stools whereas cyst predominates in non-diarrheal stools.

11. Life Cycle
• Ingestion of a mature quadrinucleate infective
cyst in contaminated food or drink and also by
hand to mouth contact.
• It is then passed unaltered through the
stomach, as the cyst wall is resistant to gastric
juice.
• In terminal ileum (with alkaline pH),
excystation takes place.
• An amoeba with four nuclei emerges and
divides by binary fission to form eight
trophozoites.
• Here they grow and multiply by binary fission.
• Trophozoites are responsible for producing
lesions in amoebiasis.
• Invasion of blood vessels leads to secondary
extra intestinal lesions.

12. • A certain number of trophozoites
come from tissues into lumen of
bowel and are first transformed into
pre-cyst forms.
• Pre-cysts secret a cyst wall and
become a uninucleate cyst.
• Eventually, mature quadrinucleate
cysts form due to increased
resistance by the host. These are the
infective forms.
• Both mature and immature cysts
may be passed in faeces.
• Immature cysts can mature in
external environments and become
infective act as carriers and spread
infection.

13. • In 10% of infected persons, amoeba
invades the host tissue, common
during the first 4 months, or short as
7 to 10 days.
• Trophozoites invade tissue after
penetrating through the intestinal
mucosa.
• This condition is self-limiting, but its
course duration is 2 to 3 yrs upto 10
yrs.
• Recovering persons from dysentery,
pass cyst in the stool called as
convalescent carriers.

14. Pathogenesis
1. Non-invasive infection:
• In many cases, the trophozoites remain confined to the intestinal lumen of
individuals - Asymptomatic carriers and cysts passers.
• Avirulent strains of Entamoeba histolytica remain in the lumen of the colon and
cause no harm.
2. Intestinal disease:
• In some patients, the trophozoites invade the intestinal mucosa.
• Those that are virulent attack and ingest the epithelial cells lining the gut wall and
then proceed to spread through underlying layers.
3. Extra-intestinal disease :
Trophozoites reaches through the bloodstream, invade extraintestinal sites such as
the liver, brain, and lungs, with resultant pathologic manifestations.

15. • The trophozoites invade the colonic
epithelium and secrete enzymes that
cause localized necrosis. Little
inflammation occurs at the site.
• In the process of invasion, flask-
shaped ulcers are formed
widespread with consequent
bleeding over large areas of the
intestine
• Progression into submucosa leads to
invasion of the portal circulation by
the trophozoites.
• Trophozoites carried by the portal
vein from the base of amoebic ulcer
in the large intestine to the Liver
• Flask shaped Ulcer

16. • Trophozoites multiplies inside liver cells and produce thrombosis of the portal
venules resulting in Anaemic necrosis of the liver cells
• Progressive destruction of concentric layers of liver cells occurs.
• Liver abscess is formed can become extensive and produce copious amounts of
purulent exudate (i.e. a thick fluid containing white blood cells, cell debris, and dead
and dying cells) resembles chocolate sauce or anchovy-paste.
• Depending on the site of the abscess, it can drain into the peritoneal cavity or the
lungs – in which case it may be coughed up.
• Right-lobe abscesses can penetrate the diaphragm and cause lung disease called
Pulmonary Amoebiasis, often results from the extension of a pre-existing hepatic
infection
• Other metastatic lesions:
• Cerebral amoebiasis
• Amoebic pericarditis
• Cutaneous amoebiasis
• Splenic abscess
• Most cases of Ameobic liver abscess occur in patients who have not had intestinal
amebiasis.

17. Clinical Findings/ Symptoms
• Acute Intestinal Amoebiasis
• dysentery (i.e. bloody, mucus containing diarrhoea)
• lower abdominal discomfort,
• flatulence
• Chronic amoebiasis: low-grade symptoms such as occasional diarrhoea,
weight loss, and fatigue also occurs.
• Amoebic abscess of the liver is characterized by weight loss, fever, and a
tender enlarged liver and right upper quadrant pain.
• Roughly 90% of infected individuals have asymptomatic infection but they may
be carriers
• Amoeboma, a granulomatous lesion may form in the cecal areas of the colon
in some patients. These lesions resemble an adenocarcinoma of the colon and
must be distinguished from them.

18. Laboratory Diagnosis
1. Intestinal Amoebiasis
• rests on finding either trophozoites in diarrheal stools or cysts in formed
stools.
• Diarrheal stools should be examined within one hour of collection to see
the ameboid motility of the trophozoite.
• The trophozoite characteristically contains ingested red blood cells.
2. Extraintestinal Amoebiasis
• Diagnosed by the use of scanning procedures for liver and other organs.
• Specific serologic tests, together with microscopic examination of the
abscess material can confirm the diagnosis.

19. 1. MICROSCOPY
• Macroscopic appearance of stool:
• Offensive dark brown semisolid stool, mixed with blood, mucus and much fecal matter.
• Microscopic examination:
• Presence of Charcot-Leyden crystals. Scanty cellular exudates, and consists of only the nuclear masses
(“pyknotic bodies).
• E. histolytica infection is distinguished from Bacillary dysentery by the lack of high fever and absence
PMN leukocytosis.
• Trophozoite nucleus has a small central nucleolus and fine chromatin
granules along the border of the nuclear membrane. The nuclei of other amoebas
are quite different.
• Trophozoites of Entamobea dispar, a nonpathogenic species of Entamoeba,
are morphologically indistinguishable from those of E. histolytica
Cyst size and the number of its nuclei.
Mature cysts of E. histolytica are smaller than those of Entamoeba coli and contain four nuclei,
whereas E. coli cysts have eight nuclei.
Concentration techniques – formal ether and ZnSO4 flotation are used if number of cysts is few.

21. 2. Culture
• A special culture medium known as TYI-S-33 supports the growth of E.
histolytica in culture. TYI-S-33 medium was developed for axenic culture
of Entamoeba histolytica (DIAMOND et al. 1978). This medium, supplemented
with bovine bile.
3. Serological Tests
• useful for the diagnosis of invasive amebiasis
• To detect anti-amoebic antibodies in invasive amoebiasis
• CIE, Gel diffusion precipitation, Indirect immunofluorescence, ELISA used
3. Imaging
MRI and ultra-sonography, CT scans detect abscess in the liver
4. Liver Aspiration
Aspirations from the amoebic liver abscess to detect trophozoites by wet mount.
5. Detection of the nucleic acid of this protozoan parasite by PCR based assays

23. • Entamoeba dispar
• Entamoeba dispar is
morphologically indistinguishable
from Entamoeba histolytica
• same life cycle but it is non-
pathogenic.
Entamoeba gingivalis
• This amoeba is commonly found in
swab samples taken from the
gingival crevices of the human
mouth.
• It does not form cysts
• Transmission is through kissing or
sharing food.

24. Treatment
• Amoebiasis is treated with metronidazole (750mg for 10 days) followed by
iodoquinol (650mg for 20 days)
• Asymptomatic carriage can be eradicated with a full course of therapy of
luminal agents like iodoquinol, diloxanide furoate (500mg for 10 days), or
paromomycin should follow
• The Cysticidal agents are commonly recommended for asymptomatic carriers
who handle food for public use.
• Metronidazole and chloroquine can be used for the treatment of extra-
intestinal amoebiasis.

25. Entamoeba coli
• Entamoeba coli the life cycle stages include; trophozoite, precyst, cyst.
• Typically the movements of trophozoites are sluggish, with broad short
pseudopodia and little locomotion.
• However, the cysts are remarkably variable in size. Entamoeba coli is
transmitted in its viable cystic stage through faecal contamination.
• Ε.coli as a lumen parasite is non-pathogenic and produces no symptoms.
• The mature cyst (with more than four nuclei) is the distinctive stage to
differentiate E.coli from the pathogenic E. histolytica.
• Specific treatment is not indicated since this amoeba is non-pathogenic.
• The presence of E.coli in stool specimen is evidence for faecal
contamination.
• Prevention depends on better personal hygiene and sanitary disposal of
human excreta.

26. Prevention
• Introduction of adequate sanitation measures
• Education about the routes of transmission.
• Avoid eating raw vegetables grown by sewerage irrigation and night
soil
• Asymptomatic carriers may excrete upto 1.5 crore cysts per day
• Proper disposal of sewage prevent transmission
• Good personal hygiene
• Washing of fruits & vegetables before consumption
• Protection of food from flies & cockroaches.
• Filtration and boiling of drinking water as the cyst is resistant to
chlorination.

27. References
• Medical Parasitology – Rajesh Karyakarte
• https://microbenotes.com/category/parasitology/
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