MODULE IN CLINICAL PARASITOLOGY (LECTURE)

TOPIC: “AMEBOID GROUP”

Lesson Title: “Amebic Colitis”

I. OBJECTIVES:

This module aims to provide an understanding of the protozoan amoeba, with emphasis on the
clinically significant species.

Specifically, at the end of the module, students are expected to be able to:
a. recall the taxonomic classification of amoebae group
b. diistinguish the pathogenic amoeba from the non-pathogenic species.
c. iIllustrate the developmental stage or life cycle of Entamoeba histolytica
d. recognize the infective stage of E. histolytica and discuss its modes of transmission;
e. identify the free-living amoeba and how humans can get infected with these protozoans
f. discuss the pathophysiology and symptomatology of pathogenic amoeba
g. enumerate some preventive measure on how to control amoebiasis.

II. INTRODUCTION:
Taxonomic Classification

Phylum Sarcomastigophora

Subphylum Sarcodina

Superclass Rhizopoda

Class Lobosea

Order Euamoebida Order Schizopyrenida Order Amoebida

Non-pathogenic: Pathogenic: Naegleria Acanthamoeba Balamuthia

Entamoeba
Entamoeba coli
histolytica
Entamoba hartmani
Entamoeba dispar
Entamoeba gingivalis
Endolimax nana
Iodamoeba butschlii
Ameobae are known for their pseudopodia, which they use for both locomotion and feeding.
Flagella, when present, are usually restricted to developmental or other temporary stages.
Because of the extension and retraction of temporary pseudopodia they do not have constant
shape.
Six species of amoebae are commonly found in the human mouth and intestines. These include
Entamoeba histolytica, E. hartmani, E. coli, E. gingivalis, Endolimax nana, Iodamoeba butschlii. Of
these only E. histolytica is of medical importance. However, other intestinal amoebae are of
interest primarily because they may be difficult to distinguish from those of E. histolytica by light
microscopy.
Amoebiasis , the disease caused by E. histolytica, is worldwide but more common in tropical and
subtropical countries. It is the third leading parasitic cause of death after malaria and
schistosomiasis. It has an annual mortality rate of 40,000 to 110,000 and infects 480 million or
12% of the world population.

High risk groups include:

– travellers
– immigrants and migrant workers
– the immunocompromised
– sexually active male homosexuals
– those in mental institutions, prisons and day -care centers

Environmental factors which determine endemicity include:

– Low socio-economic conditions
– Unsanitary excreta disposal
– Pollution of drinking water supply
– Use of night soil as fertilizer
– Food is not protected from mechanical vectors
– Contaminated food by food handlers

III. *PRE-QUIZ/ Activity:

Before we go further, try to answer the questions under Quizzes in Canvas regarding Introduction
to Protozoa.

IV. General Characteristics of Amoebae:

Genera – basis of classifying is the difference in the nuclear structure.

1. Entamoeba – nucleus has a delicate nuclear membrane and small karyosome; presence of
chromatin granules lining the nuclear membrane.
E. histolytica, E.coli, E. gingivalis, E. hartmanni, E. dispar

2. Endolimax – has a large, irregular karyosome, from this karyosome will arise fibrils which
will anchor the nuclear membrane.
Endolimax nana
 3. Iodamoeba – pearly like karyosomes, surrounded with achromatic granules, refer to peri-
endosome; achromatic because they do not absorb stain or color. In stained specimens, it
will give a halo look to karyosome.
Iodamoeba butschlii

Cyst – infective stage of amoeba

Trophozoite – vegetative stage of parasite which is the feeding stage; active, motile stage.

As a general rule:
All amoebae develop a cyst except E. gingivalis.
All amoebae are found in the colon except E. gingivalis which is an oral amoeba.
All amoeba are commensals except E. histolytica (only pathogenic intestinal amoeba)

Entamoeba histolytica

Habitat: Trophozoites reside in the mucosa and submucosa of large intestine of man.
Morphology:
Cyst = spherical, diameter ranges between 5.0 – 20 µm with a distinct cyst wall
- large race histolytica = diameter is greater than 10µm
- small race histolytica = diameter is less than 10 µm
Entamoeba hartmanni – small race E.histolytica
- number of nuclei = 1 - 4
- 4 nuclei = mature cyst ; immature cyst = 1 or 2 nuclei
- Quadrinucleated cyst which is the infective stage
- chromatoidal bars = food reserve of the parasite
- provided with rounded ends = cigar-shaped
Cysts are present only in the lumen of the colon and in formed feces.
The thick chitinous wall makes it highly resistant to the gastric acid, adverse environmental
conditions and the chlorine concentration found in potable water.

Trophozoite = motile, irregularly shaped because of the pseudopodia; measures 10 – 60 µm

(average 20-30 µm) in diameter.
- the cytoplasm can be divided into a clear outer ectoplasm and an inner finely granular
endoplasm in which RBCs, leucocytes tossue debris are found within the food vacuoles.
- is clean looking because of the presence of RBC, compared with E. coli.
RBCs may, however, be absent if infection is confined to the gut lumen.
Nucleus: spherical in shape varying in size from 4-6 µm in diameter.
= In stained preparation, shows dot-like karyosome surrounded by a clear halo (bull’s eye
karyosome).
= nuclear membrane is delicate and is lined by a single layer of fine chromatin granules.
= the space between the karyosome and the nuclear membrane is traversed by linin
network (achromatic fibrils) having spore-like radial arrangement.
 - Trophozoite is the only form present in the tissues. It usually appears only in diarrheic
feces in active cases and survives only a few hours.
a. trophozoite may eventually revert back to cyst
b. trophozoite may remain as trophozoite
= there is no proper reabsorption of water
= trophozoites survive in wet environmental condition:
1.) diarrhea = dysentery (with blood)
2.) ulcers = flask shape
3.) extra-intestinal = liver, lung, brain (rare), spleen
=where amoebic abscess occur

LIFE CYCLE
Cysts are resistant and enable amoebae to survive in the environment.

Two types of Intestinal Amoebiasis:

1. Asymptomatic 2. Symptomatic

*Incubation period: 1 – 4 weeks

*Amoebic dysentery or Amoebic liver abscesses = not synonymous with amoebiasis. It is limited to
symptoms and pathology of the infection.

Diagnosis:

Intestinal Amoebiasis:
1. Stool = direct fecal smear and look for trophozoite in watery stool, cyst in formed stool
❖ Staining technique:
For trophozoite = Quensel’s methylene blue
 For cyst = Lugol’s iodine
❖ Concentration technique:
Cyst = FECT (Formalin Ether Concentration Technique)
❖ Culture:
Locke’s egg serum (LES), Rice Egg Saline medium (RES)
Polyxenic media:
Boeck and Drbohlav’s diphasic medium (first medium in 1925)- egg slant base with
an isotonic overlay.
All these liquid media were described by Balamuth (1946) and Nelson (1947). In all
these media, it is necessary to add certain associates such as enteric bacteria or the
flagellate (Trypanosoma cruzi), as well as starch or rice flour for the amoebae to
grow and multiply.
Axenic medium: developed by Diamond in 1961; without bacteria
▪ consists of trypticase, ox-liver digest, glucose, cysteine ascorbic acid and
salts supplemented with horse serum and a vitamin mix.
▪ Yields 100 – 150 million E. histolytica from an inoculums of 10 million
amoebae.

2. Blood examination: moderate leukocytosis

3. Serological Test for intestinal and extraintestinal
- Indirect hemagglutination (IHA)
- Indirect Flourescent Antibody (IFA)
- Enzyme-linked immunosorbent assay (ELISA)

Hepatic Amoebiasis:
1. Diagnostic aspiration: Trophozoites of E. histolytica may be demonstrated by microscopy of
the pus aspirated by puncture of amoebic liver abscess in less than 15% cases.
2. Liver aspirate = wet smear, culture
- stained = Iron Hematoxylin technique
4. Blood examination: leukocytosis (15,000 – 30,000/µl of which 70-75% are PMNs)
5. Stool examination – less than 15% cases of amoebic hepatitis can be demonstrated in the
stool, which indicates persistence of intestinal infection.
6. Serological tests
7. Molecular method:
DNA probes and PCR (stool and liver aspirate) – with 87% sensitivity

NON-PATHOGENIC AMOEBAE:
These amoebae do not appear to cause disease, Knowledge of these species is of value in
differentiating the harmless commensals from the potentially pathogenic E. histolytica.

Entamoeba coli
o Worldwide parasite; lives freely in the lumen of man and is non-pathogenic.
 o Like E. histolytica, it exists in three stages: Trophozoite, precyst, and cyst.

COMPARISON BETWEEN Entamoeba histolytica and Entamoeba coli

I. CYST
1. size 5 – 20µm 10 - 33 µm
2. number of nuclei 1–4 1 - 8; 16 – 32
3. nuclear membrane Thin Thick
4. chromatoidal bars Cigar-shaped rounded ends Splintered broomstick

II. TROPHOZOITE
1. size 10 – 60 µm 15 – 50 µm
2. pseudopodium Finger-like Blunt and rounded
3. karyosome Centrally located Eccentric location
4. motility Progressive and directional Sluggish and non-
directional
5. cytoplasm Clean looking Dirty looking (viscous)
due to vacuoles because
of ingested bacteria,
yeasts and food particles

Entamoeba gingivalis

o First parasitic amoeba to be recognized in 1849 (Gros)

o found in oral cavities/ soft tartar between the teeth
o occurs as trophozoite
o Trophozoite measures 10-45µm in diameter
o Actively motile by multiple pseudopodia
- Cytoplasm: clear ectoplasm and granular endoplasm with ingested leucocytes and
epithelial cells within food vacuoles, at time bacteria and rarely RBCs.
- Nucleus: spherical, 2-4 µm; centrally located karyosome and
= nuclear membrane is lined with closely packed chromatin granules.
o inflammation of the gum, pyorrhoea alveolaris; unhygienic mouth and oral plates
o Transmission: person-to-person by close contact like kissing or from contaminated drinking
utensils.

Entamoeba dispar
o First described by Brumpt in 1925
o Formerly thought as the non-invasive non-pathogenic strain of E. histolytica
o Cyst is morphologically identical with E. histolytica
o Differs from E. histolytica in their genomic DNA and ribosomal RNA

Entamoeba hatmanni
o Cosmopolitan in distribution
o Morphologically similar with E. histolytica but both its troph and cyst are smaller
o Trophozoite has no ingested RBC
o Troph : 4-12 µm
o Cyst: 5 – 10 µm
 Endolimax nana
o The smallest amoeba (“nana” = small)
o Trophozoites are small as red blood cell which is 6 – 15µm
o Movement of trophozoite = sluggish by means of small, blunt, hyaline pseudopodia
o Cytoplasmic inclusions contain bacteria, small vegetable cells, and crystals but never RBCs.
o Nucleus =small, spherical with large irregular, eccentric karyosome;
= achromatic strands extend to the nuclear membrane
o Cyst is subspherical/ oval (8-10 µm).
Cyst when mature is quadrinucleated, with
eccentric large karyosomes, giving it a
characteristic cross-eyed appearance.

Iodamoeba butschlii / I. williamsi

o Trophozoite: 6-20 µm
- 6– 20µm; fairly active in freshly evacuated unformed stool and show sluggish movement
in older stools
- Endoplasm contains coarse and fine granules and has bacteria and yeast cells in food
vacuoles.
- Glycogen vacuole may be occasionally seen, which stains golden brown with Iodine.
- uninucleated with large centrally located karyosome
o Cyst
- 8 – 15µm; ovoid or pyriform in shape
- uninucleated with large centrally located karyosome
- No peripheral chromatin lining.
- Cytoplasm contains large glycogen vacuole.
- has very big glycogen mass
- Glycogen mass develop in golden brown color in iodine solution which therefore refer to
as Iodine cyst.

V. Additional Readings and References:

Related videos/ websites:
*Uploaded Files: (see Files under Canvas)
Entamoeba histolytica
Non-pathogenic Amoebae
VI. *Self-assessment Activity (Canvas)

REFERENCES:

Belizario, Vicente Y. Jr & Solon, Juan Antonio A. PHILIPPINE TEXTBOOK OF MEDICAL PARASITOLOGY.
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Edition. Philadelphia: Lea & Febigez.
Brown, Harold W. and Neva, Franklin A. BASIC CLINICAL PARASITOLOGY. United States of America:
Appleton & Lange.
Garcia, Lynne Shore DIAGNOSTIC MEDICAL PARASITOLOGY 5th Edition. ASM Press.
Heelan, Judith S and Ingerson, Frances W. ESSENTIALS OF HUMAN PARASITOLOGY, 4TH Edition.
Delmar Cengage Learning, 2001.
John, David T., Edward K. Markell and Voge’s MEDICAL PARASITOLOGY; 9th Edition. Philadelphia:
Elsevier Saunders, c2011.
Henry,John Bernard CLINICAL LABORATORY DIAGNOSIS and MANAGEMENT BY LABORATORY
METHODS. Philadelphia: W.B. Saunders Company, c2017.
Roberts, Larry S.and John Janovy Jr. Larry S. Roberts’ FOUNDATIONS OF PARASITOLOGY; 8TH Edition.
New York: McGrow Hill Science/Engineering/Math. c2005.