2. OBJECTIVES
1. Name the common intestinal amoebae &ciliates that infect
humans
2. Of the intestinal amoebae, name the organisms that are
pathogenic to human
3. Outline the life cycle of Entamoeba histolytica /Balantidium coli
indicating the stages that cause pathogenic effects and are of
diagnostic importance in the above
4. Identify points in Life cycle where preventive measures are
applicable
5. Describe the mechanism of pathogenesis
6. Describe the pathogenesis and clinical features of these stages
7. Describe the mode(s) of transmission, prevention and control of
amoebiasis
8. Describe the laboratory methods of diagnosis of these
organisms
3. Intestinal protozoan
• 1.Amoebae – moves by means of pseudopodia
• 2. Ciliates – are propelled by rows of cilia that
beat with a wave like motion
3. Flagellates- move by long whip like flagellae
4. Coccidia: lack the specialized organelles of
motility
Phylum protozoa is classified into 4
subdivisions based on methods of locomotion
6. can exist as trophozoite (growing stage) or cyst
( dormant stage)
Differentiate on morphological features of either
trophozoite or cyst
Differentiating features of trophozoite:
Size,
Type of motility – directional or non- directional
fast or sluggish
character of pseudopodia
,Cytoplasmic inclusion bodies : Red blood cells,
food vacuoles containing bacteria, yeast
7. Differentiating features of cyst :
size
shape
number of nuclei, structure of nuclei
presence of glycogen mass
Chromatoid body or bar - coalesced RNA
within the cytoplasm
number of nuclei, arrangement of peripheral chromatin,
position of the karyosome
Nuclear structure:
Chromatin ; Nuclear DNA present as peripheral
chromatin
Karyosome: small condensed mass of chromatin within the
nuclear space
Peripheral chromatin – chromatin adhering to nuclear
membrane
8. Genus : Entamoeba
Parasites of alimentary tract - man, monkeys
vertebrates and invertebrates
Characteristics of this genus :
Nucleus more or less spherical
Nuclear membrane line with chromatin
granules
Small karyosome situated at or near the
centre
Trophozoite has single nucleus
10. Amoebae that parasitize humans
Intestinal amoebae: ( inhabit the large intestine)
Entamoeba histolytica
E.dispar
E.coli
E.hartmani
Endolimax nana
Iodamoeba butschlii
Dientamoeba fragillis
Oral cavity : Entamoeba gingivalis
11. There are two stages in the life cycle of
these amoebae.
1.Trophozoite:mortile and feeding
stage. Multiply by binary fission
2. Cyst : Inactive, non motile and
infective stage
No cyst stages in D.fragilis &
E.gingivalis
12. Of several species of amoebae live in the
alimentary tract of human MAJORITY are
commensals ONLY Entamoeba
histolytica is pathogenic D.fragilis and I.butschlii,
may cause intestinal infection
14. • cosmopolitan distribution
• worldwide incidence: 0.2-50%
• highest prevalence in areas with
poor sanitation
• no animal reservoirs
•estimated 50 million cases/year
100,000 deaths/year
Entamoeba histolytica
15. Disease: amoebiasis
Blood and mucous diarrhoea
Pathogenic organism parasitize large
intestine of man
E. dispar identical morphology but not
Invasive ( non-pathogenic)
16. RBCs
Nucleus
20-40 µm, motility-active, progressive,
directional
Pseudopodia- finger like, hyaline, very rapidly
extruded
Inclusions- red blood cells (invasive forms)
Nucleus- single, fine central kayosome,
regular peripheral chromatin
Trophozoite
17. Cyst – spherical, 10-20 µm (E. hartmanni <10 µm)
Nuclei: 1-4, structure like in trophozoite
Chromatoid bodies: thick, 1-2 stain like chromatin,
disappear as cyst matures (does not stain with Iodine)
E. dispar identical morphology
18. Life cycle
Infective stage
• Ingestion of mature
cysts
• Excysts in small
intestine
• Each cyst give rise
immature trophozoites
• Maturation takes place
in caecum
•Trophozoites feed grow
and divide causing
pathological effects
19. Amoebiasis
Pathogenesis - Infection with
E.histolytica does not
necessarily lead to disease. The
outcome depends on :
•Host factors
•Parasite factors
20. Host Factor Contributions
• Physico-chemical environment of the
gut influenced by bacterial flora, mucus
secretion & gut motility
• Degree of immunological resistance
21. Important virulence factors of E.histolytica
• Adhesion molecules ( N- acetyl-D-
galactosamine inhibitable lectine
Gal/GalNac) – adhesion to colonic mucine and host
cells
• induce contact dependent cytolysis,
• Channel-forming peptides(Amoebapores):
Stored in cytoplasmic granules & release
following target cell contact, forms iron
exchanging channels in plasma membrane –
lysing the target cells
Parasitic factors
22. 3. Cystein protinases –
Aid in penetration of host tissue by digesting
extracellular matrix, cleaving collagen, elastin,fibrinoge in
extracellular matrix by stimulating host cell proteolytic cascade
Resistance to host response
• complement resistance-inactivates theinactivates the
complement factorscomplement factors and are thus resistantand are thus resistant
to Complement mediated lysis.to Complement mediated lysis.
• Limit the effectiveness of humoral
response by degrading
both IgA and IgG
4. Species/strain differences; E. dispar non
invasive, Pathogenic zymodemes =E.histolytica
24. • Penetration of mucus layer
• contact-dependent killing of epithelium
• breakdown of tissues (extracellular matrix)
• contact-dependent killing of neutrophils, leukocytes, etc.
initially produce focal and superficial erosions in large
intestine with unaffected mucosa in between
Adhere to colonic mucin
and host clls
26. •Trophozoites advance laterally and downward
into the submucosa producing a 'flask-shaped'
ulcer ( typical appearance of intestinal
amoebiasis)
Flask shaped ulcers -Base in submucosa and small opening on
the mucosal surface
• Trophozoite penetrates the intestinal
epithelium and then the muscularis mucosa &
enter in to submucosa
27. Trophozoites penetrate the muscle
and serous layers leading to
intestinal perforations ,peritonitis
Rarely involvement of blood vessels at the base of the
ulcer may produce profuse bleeding
Amoeboma - Amoebic granuloma
An inflammatory thickening of the intestinal wall,
due to repeated invasion of colon by E histolytica
common sites- ascending colon & caecum
Haematogenous spread to other organs
35. Clinical features
Intestinal disease
Majority of infections are asymptomatic
[cyst passers are infective carriers]
asymptomatic cyst passer
• Amoebic colitis
Gradual onset ( symptoms presenting over 1-2
weeks)
abdominal pain, tenesmus , watery or bloody
diarrhoea, anorexia, loss of weight. Fever only
10- 30%
Rectal bleeding without diarrhea can occur,
especially in children
38. Extraintestinal Disease _ sings &
symptoms depend on the organ affected
liver abscess –
Frequently affect adults than children,
Male>female
60-70% of patients with amebic liver abscess do
not have concomitant colitis, a history of dysentery
within the previous year
hepatomegaly, liver tenderness, pain in the
upper abdomen, High fever and anorexia,
Weight loss, vomiting, fatigue
40. Trophozoites
Direct wet faecal smears in saline can
demonstrate motile trophozoite. Fresh sample of
faeces ( preferably with in 30 min) should be
examine to visualize live trophozoite.
confirmed on a permanently stained smear to
identify morphological features of nucleus
Eg; Trichrome or Iron haematoxylin
• Biochemical Methods: Culture and
Isoenzyme analysis to differentiate
E.dispar from E.histolytica
44. Cyst
Wet faecal smear ( saline or iodine)
If cysts are few to be present in direct
smear, cysts can be concentrated
either by floatation ( Zinc sulphate
centrifugal floatation) or by
sedimentation ( Formal-Ether )
Faecal concentration methods
46. E.Coli cyst
Size – 10 -20 µm, >4nuclei
Nucleus ; eccentric karyosome with
irregular coarse chromatin
Chromatoid bodies infrequent ,needle
shape when present
Differentiation of E.Hislolytica from other non-
pathogenic intestinal protozoa is very important
48. Acute dysentery- predominant form
trophozoites
saline, stained smear, culture
Colitis – cysts - saline, iodine, concentration
methods
Faecal examination: minimum of 3 samples in
7 days
wet/permanent/culture
49. Diagnosis – Intestinal amoebiasis
Definitive diagnosis
[GOLD STANDARD]
– demonstrate parasite in
stools/rectal smears
STOOL
FULL REPORT
= SFR
Trophozoites
with ingested
red blood cells indicate
invasive amoebiasis
Presence of cysts does
not indicate active
disease but infective
carriers
(cysts are infective)
Without the specific
presence
of ingested RBCs in the
cytoplasm
the pathogen, E. histolytica
& the non pathogen, E.
dispar
Are morphologicaly
identical BUT
Biochemically different
50. Detection of E.histolytic specific antibodies
By Enzyme linked immunosorbent assy(ELISA)
Useful in non-endemic areas where E. histolytica
infection is not common
Antigen Detection in stool
• Antigen-based ELISA s
Advantages
Differentiate E. histolytica from E. dispar; (ii) they have
excellent sensitivity and specificity;
Immunodiagnosis
51. Emerging methods in Diagnosis
• These are considered the
most useful tests for
detecting E. histolytica.
They test directly for the
parasite itself by exposing
some stool to a strip of
paper coated with
antibodies. The parasites
will stick to the antibodies
on the paper. The test
distinguishes E.
histolytica from other
parasites.
• Disadvantage : costly
52. Molecular Biology-Based Diagnostic
Tests - PCR
• Detection of parasite DNA in faeces by PCR
• Provide high sensitivity and specificity for the
diagnosis of intestinal amoebiasis
54. •abscess aspiration
only selected cases
reddish brown liquid
trophozoites at the abscess wall
•imaging
X –ray, CT, MRI,
ultrasound
•Abscess fluid Ag detection (ELISA)
55. Typical aspirate- chocolate syrup
Trophozoites
are found on marginal wall
Commonly found in the last portions of
aspirated material
56.
57. Peters & Gilles. Atlas of Tropical Medicine and Parasitology- 4th
Ed. Mosby-Wolfe 1995
CT scan of abscess in R lobe
X ray showing fluid level
59. Test
Colitis Liver abscess
Sensitivity Specificity Sensitivity
Microscopy
(stool)
<60% 10-50% <10%
Microscopy
(abscess fluid)
NAb
NA <25%
Stool antigen
detection
(ELISA)
>95% >95% Usually negative
Serum antigen
detection
(ELISA)
65% (early) >90% ∼75% (late),
100% (first 3∼
days)
Abscess
antigen
detection
(ELISA)
NA NA ∼100% (before
treatment)
PCR (stool) >70% >90% Not done
Serum
antibody
detection
(ELISA)
>90% >85% 70-80% (acute),
>90%
(convalescent)
sensitivity and specificity of tests of
diagnosis for amoebiasisa
60. Transmission
Through cysts
Sources of infection:
Food and water contaminated with infected
faeces.
Food handlers excreting cysts are an
important source of contamination of foods
Houseflies also act as a mechanical vectors
contaminating food
Sexual transmission
• Direct – hand to mouth
• Indirect- contamination of food/water
61. Man is only reservoir host
Because of the protection conferred by
cyst wall , cyst can survive days and
weeks in external environment
Cyst Can be killed:
Boiling- Above 68 ° C
Iodine (200 ppm)/acetic acid 5-10%
Remove from water by sand filtration
Ordinary chlorination does not kill cysts
62. Epidemiology
Amoebiasis is cosmopolitan but no correlation
between infection and disease
Generally in developed countries asymptomatic
In tropics/low socio-economic standards
High pathogenicity
High risk groups: travelers, institutional inmates
homosexuals,
immunocompromised individuals, children in day
care centers
64. Food safety
• Thoroughly cook all raw foods.
• * Thoroughly wash raw
vegetables and fruits before
eating.
• * Reheat food until the internal
temperature of the food
reaches at least 167º.
• Wash your hands before
preparing food, before eating,
after going to the toilet or
changing diapers
66. What are ciliates ?
Protozoa with cilia
Cilia -
Hair like structures used for locomotion
and feeding.
Shorter than flagella and more in
number
67.
68. • Use cilia for movement or feeding
• Can have more than one nucleus
(macronucleus, micronucleus)
• Feed through a “mouth” like structure (oral
groove,
Ciliophora – ciliates
69. Generally larger than other protozoa
Reproduce by binary fission
ONLY ciliate that is known to parasitize
man is Balantidium coli
70. Balantidium coli
Largest protozoan parasite of man
A common parasite of pigs
Pig the main reservoir
Human infection is less frequent
Parasitize distal ileum and colon
Invade the mucosa and causes blood
and mucous diarrheoa
It is a zoonotic infection
71. C/f similar to amoebic dysentery
but no extra-intestinal spread
Pathogenic to man as it invade the
intestinal tissue
78. Regarding E. histolytica
A. Cyst is the infective stage
B. Does not attach to intestinal mucosa
C. Inhabits the human large intestine
D. Extaintestinal spread is possible
E. Nucleus has a central karyosome
Regarding amoeba
A. E. gingivalis has cyst stage in their life cycle
B. Can differentiated by their characteristic movements
C. E. dispar is a human pathogen
D. E. coli and E. histolytica are morphologically identical
79. Regarding Balantidium coli
A. It is not pathogenic to human
B. Trophozoite has only one nucleus
C. It is a zoonotic parasite
D. Cyst is covered with cilia
E. Trophozoite is the infective stage to human
True /false E.histolytica
Inhabits human large intestine
E. Histolytica cyst is a infective stage to human
Transmitted by faeco-oral route
E.Histolytica trophozoite is morphologically identical to E. dispar
80. True or false
Genus Entamoeba has large katyosome in side the nucleus
E. Histolytica trophozoite moves sluggishly
E. Histolytica trophozoite has single nucleus with centrally placed karyosome
E. Gingivalis has trophozoite and cyst in their life cycle
Acute amoebic dysentery, predominant form is cyst in stools
Flask shaped ulcers are typical lesion in intestinal amoebiasis
Trophozoites in faecal samples is a commonly associated with hepatic amoebiasis
In amoebic colitis, predominant form in the faeces is trophozoite
E histolytica and E dispar cysts cannot differentiate microscopically
Fever is a common clinical feature of amoebic colitis.
Abscess fluid microscopy is useful in the diagnosis of amoebic liver abscess
