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Introduction
Disease biology
Epidemiology
Clinical
Summary
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Amoebiasis
E-Learning Module
Matt Pugh
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begin
Introduction
Disease biology
Epidemiology
Clinical
Summary
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Introduction
Welcome to the amoebiasis e-learning module. Amoebiasis is a
parasitic protozoan disease that affects the gut mucosa and liver,
resulting in dysentery, colitis and liver abscess. The causative
agent, Entamoeba histolytica, is a potent pathogen that is spread via
ingestion of contaminated food and water. Globally, amoebiasis is
highly prevalent, and is the second leading cause of death to
parasitic disease.
This resource will outline the disease biology, epidemiology and
clinical principles of amoebiasis.
Introduction
Disease biology
Epidemiology
Clinical
Summary
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Learning Outcomes
The learning outcomes for this moudule are:
• Understand the biology, life cycle and mode of transmission of the amoebiasis
causative organism, Entamoeba histolytica.
• Understand the pathological processes that lead to disease in amoebiasis
• Know the distribution of amoebiasis worldwide, and which geographical,
cultural and economic factors can pre-dispose to the disease
• Know how the disease presents according to the infected organ system.
• Be able to outline the treatment and management of symtomatic and
asymtomatic patients.
• Outline the key features of the developing gal-lectin vaccine.
Introduction
Disease biology
Epidemiology
Clinical
Summary
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How to use this module
The module is split into five main sections: introduction, disease biology,
epidemiology, clinical and summary. The information required to complete the
learning outcomes are contained within the disease biology, epidemiology and
clinical sections. At the end of each of these sections there will be a self
assessment section. You will require a pen and paper to write down your answers.
How to navigate
Navigate through the module using the
blue arrows to go back and forth. You may
skip straight to, or back to a section by
clicking the tabs at the top of screen.
Additionally, you may skip through the
sub-sections by clicking on the tabs at
the side of the screen.
Introduction
Disease biology
Epidemiology
Clinical
Summary
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Causative Organism
Causative
Organism
• The causitive orgainism is parasitic protazoan, called Entamoeba
histolytica.
Life Cycle and
transmission 1
•What was once thought to be a single entity, is now recognised as
two morphologically identical but genetically distinct forms; E.
histolytica (pathogen) and E. dispar (commensal).
Life Cycle and
transmission 2
•This has affected our understanding of amoeba distribution. Many
suspected cases of E. histolytica carrier, may simply have been E.
dispar colonisation
Pathogenesis 1
• The WHO recommendes that E. histolytica colonisation should be
treated, however, treatment is unnecessary for E. dispar
colonisation
Pathogenesis 2
Self Assessment
E. Histolytica
Introduction
Disease biology
Epidemiology
Clinical
Summary
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Life cycle and transmission 1
Causative
Organism
• Entammoeba histolytica has a biphasic life cycle, existing in two
forms; as an infectious cyst and an amoeboid trophozoite
Life Cycle and
transmission 1
Mouth - Cyst ingested
Excyst to trophozoite
Passed in stool
Life Cycle and
transmission 2
Amoebic disease
Pathogenesis 1
Pathogenesis 2
Self Assessment
Cyst
Trophozoite
Invades gut mucosa – cyst formation
Introduction
Disease biology
Epidemiology
Clinical
Summary
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Life cycle and transmission 2
Causative
Organism
Life Cycle and
transmission 1
Life Cycle and
transmission 2
Pathogenesis 1
Pathogenesis 2
Self Assessment
• Cysts (10-15μm) are ingested via contaminated food or water. A
refractile wall containing chitin, allows the cyst to survive stomach
acid.
• In the terminal ileum or colon, the parasite excysts and begins the
trophozoite stage.
• Trophozoites (10-50μm) are highly motile and pleomorphic. They
are unable to survive outside the human gut.
• Energy is derived from the ingestion of bacteria and food particles.
No mitochondria are present in trophozoites. Respiration enzymes
are prokaryotic in origin and are anaerobic, converting:
glucose + pyruvate
ethanol
•Trophozoites reproduce by binary fission and encyst in the colonic
wall. Cysts are passed in the stool where they become infectious.
• The signal for encystation is thought to be via epithelial
galactose/N-acetylgalactosamine specific lectin (gal-lectin)
binding protein.
Introduction
Disease biology
Epidemiology
Clinical
Summary
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Pathogenesis 1
Causative
Organism
Life Cycle and
transmission 1
Life Cycle and
transmission 2
Pathogenesis 1
Pathogenesis 2
Self Assessment
• Amoebic trophozoites invade the colon causing colitis. They may also invade the
portal circulation and travel to the liver, causing liver abscess.
Gastrointestinal Pathology
• The spectrum of colitis in amoebiasis ranges from mucosal thickening, to
multiple cyst formation, to diffuse Inflammation / oedema, to necrosis and
perforation of colonic wall.
•Binding of E. histolytica to epithelial cells via gal-lectin. This molecule shows
homologous to human CD59, conferring resistance to complement . A change in
the epithelial permeability is induced, probably via the inter-cellular tight
junctions.
• Cell lysis and apoptosis of mucosa are thought to be mediated by amoebapores,
peptides capable of forming pores in lipid bi-layers.
•Trophozoites invade through to the submucosa causing flask shaped cysts .
• Cysteine proteases released by trophozoites digest extracellular matrix in liver
and colon, and induce interleukin-1 mediated inflammation. Proteases also cleave
IgA and IgG antibodies.
•Neutrophils and macrophages are drawn to invasion sites. E. histolytica can lyse
neutrophils leading to further tissue damage, and contributing towards the
induction of diarrhoea.
•Inflammation is a significant cause of tissue damage, however, innate immunity
may be the main combatant against the disease.
Introduction
Disease biology
Epidemiology
Clinical
Summary
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Pathogenesis 2
Hepatic Pathology
Causative
Organism
Life Cycle and
transmission 1
Life Cycle and
transmission 2
• Trophozoites invading the colonic mucosa may enter the hepatic
circulation and reach the liver
•Well circumscribed abscesses are
formed in the liver containing
liquefied cells surrounded by
inflammatory cells and trophozoites
•Adjacent parenchyma is usually
unaffected
Amoebic liver abscess
Pathogenesis 1
Pathogenesis 2
Self Assessment
Histological cross section of classical flask
shaped amoebic ulcer in colonic mucosa.
Amoebic colitis with multiple ulcer formation
Introduction
Disease biology
Epidemiology
Clinical
Summary
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Questions
Causative
Organism
Life Cycle and
transmission 1
Life Cycle and
transmission 2
1) Which of the following organisms is the pathogenic causitive
agent of amoebiasis, and which is a commensal?
•
Entamoeba histolytica ………………………………………….
•
Entamoeba dispar ……………………………………………….
2) Draw a simple diagram oulining the life cycle of entamoeba
histolytica.
3) Which two organs does E. histolytica primarily invade?
Pathogenesis 1
4) What is the name and mechanism of action of the peptide
responsible for cell lysis and apoptosis in the mucosa?
Pathogenesis 2
5) What is the name of the enzyme group released by trophozoites
to digest the extra-cellular matrix
Self Assessment
Reveal
Answers
Introduction
Disease biology
Epidemiology
Clinical
Summary
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Answers
1)
Causative
Organism
Life Cycle and
transmission 1
Life Cycle and
transmission 2
Pathogenesis 1
Which of the following organisms is the pathogenic causitive agent of amoebiasis,
and which is a commensal?
•
Entamoeba histolytica ……………Pathogen…………….
•
Entamoeba dispar ……………Commensal……………….
2)
Draw a simple diagram oulining the life cycle of entamoeba histolytica.
Mouth - Cyst ingested
Excyst to trophozoite
Passed in stool
Amoebic
disease
Cyst
Trophozoite
Invades gut mucosa – cyst formation
3) Which two organs does E. histolytica primarily invade?
Colon and liver
Pathogenesis 2
Self Assessment
4) What is the name and mechanism of action of the peptide responsible for cell lysis
and apoptosis in the mucosa?
Cell lysis and apoptosis of mucosa are thought to be mediated by amoebapores,
These peptides form pores in lipid bi-layers of mucosal cells, leading to cell
leakage resulting in lysis and apoptosis.
5) What is the name of the enzyme group released by trophozoites to digest the extracellular matrix ?
Cysteine proteases
Introduction
Disease biology
Epidemiology
Clinical
Summary
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Epidemiology
Epidemiology
Susceptibility
Self Assessment
• Amoebiasis is found primarily in developing tropical and
subtropical countries where sanitation is poor, leading to a direct
link between faeces and ingestion (see Box-1). Occasionally cases
are reported in non-endemic areas e.g. UK and USA. Usually due to
travel and immigration from endemic areas.
• There are an estimated 40,000-100,000 deaths due to amoebiasis
worldwide each year.
Box-1. Amoebiasis rates/figures in
endemic regions
-Egypt: accounts for 38% of
patients presenting with acute
diarrhoea in outpatient clinic.
-Mexico:1.3 million cases
reported in 1996.
-Hue, Vietnam: 1500 of a
1million population over 5
years
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Disease biology
Epidemiology
Clinical
Summary
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Susceptibility
Epidemiology
•Generally considered to affect children and adults, of both sexes
equally. However, some data and anecdotal evidence suggests a
male predominance.
Susceptibility
•Amoebic liver abscesses are most common in males, 18-55.
Self assessment
•Susceptibility to liver abscess conferred by HLA-DR3 and
complotype SC01 in the Mexican populations
•Other risk factors include oral and anal sex, and contact with
contaminated enema apparatus.
Introduction
Disease biology
Epidemiology
Clinical
Summary
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Questions
Epidemiology
1) How many deaths are caused by amoebiasis each year?
a) 1000 – 5000
b) 40,000-100,000
c) 500,000-1,000,000
Susceptibility
2) Which part of the world is amoebiasis primarily found?
Self assessment
a) Developed countries b) Tropical and subtropical c)Cold climates
3) Does amoebiasis affect males or females more?
4) Apart from poor sanitation, what other risk factor pre-dispose to
amoebiasis infection?
Reveal
Answers
Introduction
Disease biology
Epidemiology
Clinical
Summary
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Answer
Epidemiology
1) How many deaths are caused by amoebiasis each year?
a) 1000 – 5000
b) 40,000-100,000
c) 500,000-1,000,000
Susceptibility
2) Which part of the world is amoebiasis primarily found?
Self assessment
a) Developed countries b) Tropical and subtropical c)Cold climates
3) Does amoebiasis affect males or females more?
Thought to affect both sexes equally, however, anecdotal
evidence suggests a male predominance.
4) Apart from poor sanitation, what other risk factor pre-dispose to
amoebiasis infection?
Other risk factors include oral and anal sex, and contact with
contaminated enema apparatus.
Introduction
Disease biology
Epidemiology
Clinical
Summary
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Presentation
Presentation
Diagnosis
Treatment and
Management
Vaccine
Development 1
Vaccine
Development 2
Vaccine
Development 3
Self Assessment
Some individuals carry E. histolytica asymptomatically. 4 -10% will go on to
develop the disease within a year.
Gastroenterological
• Gradual onset (weeks) of bloody diarrhoea, occasionally with small volumes of
mucoid stool. If blood is not visible, stool is usually ‘haem’ positive due to the
breach of the mucosa.
• Abdominal pain and tenderness.
• Leucocytes and pus may be present in stool. Fever present in <40% of patients.
• Weight loss and anorexia can be present.
•In more severe cases fulminant amoebic colitis develops. Liver involvement is
more common in these cases, along with paralytic ileus, toxic megacolon and
mucosal sloughing. Over 75% of patients with fulminant colitis develop intestinal
perforation.
• Local inflammatory masses, amoebomas, may cause obstructive symptoms.
Hepatic
• More common in men
• Liver abscess pan present in conjunction with bowel symptoms (10% of cases), or
in isolation.
• Sudden onset of upper abdominal pain with fever. Pain may radiate to right
shoulder or be exacerbated by repiratory movements.
• Hepatic tenderness may be present. Jaundice is unusual.
•Complicated liver abscess may develop if abscess ruptures into the peritoneal,
pericardial or pleural cavity. Morbidity and mortality is high.
•Rarely, trophozoites may also invade the respiratory tract, brain and GU tract
Introduction
Disease biology
Epidemiology
Clinical
Summary
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Diagnosis
Presentation
Diagnosis
Treatment and
Management
Vaccine
Development 1
Vaccine
Development 2
Vaccine
Development 3
Self Assessment
• Clinical history is important. In low resource settings this may be
the means of diagnosis. A good travel history is important as disease
may develop years after a visit to an endemic area.
•Demonstration of E. histolytica in stool by microscopy (old), or ELISA
assay for antigen detection. Trophozoites only survive for short
periods of time, therefore, fresh stool samples should be used
•Colonoscopy to confirm colitis and tissue biopsy for amoeba
•Liver abscess; space occupying lesion on CT/USS with positive
amoebic serology
Introduction
Disease biology
Epidemiology
Clinical
Summary
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Treatment and Management
Presentation
Diagnosis
Treatment and
Management
Vaccine
Development 1
Vaccine
Development 2
• Amoebiasis, in particular with liver involvement, can be fatal if not
treated. Chemotherapy can effectively cure ameobiasis.
• Nitroimidazole (e.g.metronidazole) is used to treat the invasive
pathogens – 800mg t.d.s for 10 days.
• This is followed by a luminal agent (e.g.diloxanide furoate) to
eliminate colonisation – 500mg t.d.s for 10 days. This is also suitable
for asymptomatic individuals.
•Complicated liver abscesses should be drained surgically.
Prevention
Vaccine
Development 3
Self Assessment
•Boiling water for at least ten minutes kills amoebic cysts
effectively. Chlorine and iodine tablets are not thought to be 100%
effective.
Introduction
Disease biology
Epidemiology
Clinical
Summary
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Vaccine Development 1
Presentation
Diagnosis
Treatment and
Management
Vaccine
Development 1
Vaccine
Development 2
Vaccine
Development 3
Self Assessment
Amoebiasis incidence could be vastly reduced with simple
sanitation and hygiene measures. However, given the current
political and economic climate, this seems unlikely in the near
future. Furthermore, with developing drug resistance in E. histolytica,
vaccine development could be effective.
Why vaccinate?
• Could prevent development of amoebic disease and associated
sequelae.
• Humans only host for E. histolytica, therefore eradication vaccine
would eliminate E. histolytica from the carrier pool.
Which target?
•A number of potential targets have been identified including
cysteine proteases, LPGs and peroxiredoxins. The two most
promising antigens identified are Serine-Rich E. histolytica Protein
(SREHP) and Galactose/N-acetylgalactosamine lectin (Gal-lectin).
Here the potential Gal-lectin vaccine will be described
Introduction
Disease biology
Epidemiology
Clinical
Summary
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Presentation
Diagnosis
Treatment and
Management
Vaccine
Development 1
Vaccine Development 2
Gal – lectin and the immune response
•Gal-lectin is a 260kDa complex protein which consists of disulphide linked light (35
kDa) and heavy subunits (170kDa). The heavy chain is cysteine rich and is thought to be a
target for immune responses, inducing a Th1 cytokine cell mediated immune response
•Macrophages induced by cytokines interferon(INF)-γ have amoebocytic activity, as do Tcells exposed to INF-γ exposed or TNF.
•Trophozoite killing by macrophages is done via nitric oxide (NO). Gal-lectin can directly
activate macrophages to release NO and induce mRNA transcription of Th1 cytokines,
thereby enhancing the cell mediated immune response.
•Monoclonal antibodies (MAbs), antiserum and IgA secreted from the gut mucosa
against the Gal-Lectin antigen, have the ability to inhibit E. histolytica adherence to
colonic mucosa in vitro.
IgA, MAb, antiserum
Macrophage
Key
Vaccine
Development 2
Vaccine
Development 3
Activate
NO
Attack
Trophozoite
Inhibit
Cytokines
e.g INF-γ
Mucosa
epithelial cell
Gal-lectin
Self Assessment
Th1 cell
Introduction
Disease biology
Epidemiology
Clinical
Summary
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Vaccine Development 3
Presentation
Diagnosis
Vaccine
Development 1
Vaccine
Development 2
Protection %
Treatment and
Management
Both Gal-lectin classical and DNA based vaccines have been tested in murine
models.
Protection conferred from purified and
recombinant vaccines
Gal-lectin classical vaccine
Purified (lectin) and recombinant galletin (LecA) have been trialled, showing
good efficacy in preventing E.
histolytica pathogenesis.
Immunisation were intra-nasal and
intra-peritoneal in order to stimulate
the gastrointestinal immunity.
Gal-lectin DNA vaccine
• DNA of heavy gal-lec subunit used as vaccine (see Fig-5)[4]
Induced Th1 mediated anti-body specific response greater than control (nothing),
however response was small. Vaccine moderately inhibited trophozoite adherence
in vitro via anti-body action.
Production of DNA gal-lectin DNA vaccine in murine model.
Vaccine
Development 3
Self Assessment
170
35
Gene
coding
for portion of
heavy gal-lec
subunit isolated
Transfected
into plasmid
Plasmid injected
intra-muscularly
Muscle cells take up
and incorporate gallec sequence in DNA
Protein expressed
and
immune
response induced
Introduction
Disease biology
Epidemiology
Clinical
Summary
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Questions
1) What are the symptoms of gastrointestinal amoebiasis?
Presentation
2) What are the symptoms of hepatic amoebiasis?
Diagnosis
3) Why is a good travel history important in diagnosis of
amoebiasis?
Treatment and
Management
4) What investigations can be performed to confirm a diagnosis?
Vaccine
Development 1
Vaccine
Development 2
Vaccine
Development 3
Self Assessment
5) Name two drugs and dosage regimes that can be used to treat
amoebiasis.
6) Is the following statement true or false?
“chlorine and iodine can be used to decontaminate water of
E.histolytica with 100% effectiveness”
7) Does Gal-lectin induce a Th1 or Th2 cell mediated immune
response?
Reveal
Answer
Introduction
Disease biology
Epidemiology
Clinical
Summary
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Presentation
Diagnosis
Answers
1) What are the symptoms of gastrointestinal amoebiasis?
Gradual onset (weeks) of bloody diarrhoea, abdominal pain and tenderness, fever present in
<40% of patients, weight loss and anorexia, amoebomas, may cause obstructive symptoms.
2) What are the symptoms of hepatic amoebiasis?
Sudden onset of upper abdominal pain with fever. Pain may radiate to right shoulder or be
exacerbated by repiratory movements.
Hepatic tenderness may be present. Jaundice is unusual
3)
Treatment and
Management
Vaccine
Development 1
Vaccine
Development 2
Vaccine
Development 3
Self Assessment
Why is a good travel history important in diagnosis of amoebiasis?
A good travel history is vital to ascertain whether a patient has visited an endemic area. The
disease may develop over a year after travel.
4)
What investigations can be performed to confirm a diagnosis?
Demonstration of E. histolytica in stool by microscopy (old), or ELISA assay for antigen detection.
Colonoscopy may be performed to check for colitis and biopsy. Check for liver abscess
with USS or CT.
5) Name two drugs and dosage regimes that can be used to treat amoebiasis.
Nitroimidazole (e.g.metronidazole)– 800mg t.d.s for 10 days. This is followed by a luminal agent
(e.g.diloxanide furoate) 500mg t.d.s for 10 days.
6)
Is the following statement true or false?
“chlorine and iodine can be used to decontaminate water of E.histolytica with 100%
effectiveness”
Boiling is the most effective methos for water decontamination
7)
Does Gal-lectin induce a Th1 or Th2 cell mediated immune response?
Th1 cell mediated response
Introduction
Disease biology
Epidemiology
Clinical
Summary
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Summary
References
Summary
•Amoebiasis is a major global cause of mortality and morbidity,
due to dysentery. The causative organism, E. histolytica.
•E. histolytica has a biphasic life cycle and exists as an infective
cyst and pathological trophozoite.
•The disease is spread via contaminated food and water, usually
due to poor sanitation.
•The disease is found in tropical and sub-tropical parts of the
world.
•Every year, 40,000-100,000 people die from amoebiasis
•Certain genetic traits pre-dispose to certain pathologies.
•Patients usually present with abdominal pain, bloody stools and
fever. Hepatic symptoms are more acute with upper abdominal
pain and radiation to the right shoulder.
•Treatment is with Nitroimidazole (e.g.metronidazole) and a
luminal agent. Spread can be prevented by boiling water.
•A potential gal-lectin vaccine is currently in development. Good
results have been yielded with native gal-lectin vaccines, and
moderate results with a DNA based vaccine. Immunity appears to
be mainly via a Th1 cell medicated response and secretory IgA
Introduction
Disease biology
Epidemiology
Clinical
Summary
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References and further reading
Summary
•Gaucher D., Chadee K. (2003). Prospect for an Entamoeba histolytica
Gal-lectin-based vaccine. Parasite Immunology. 25, 55–58 (review)
References
•Gaucher D., Chadee K. (2002). Construction and immunogenicity of a
codon-optimized Entamoeba histolytica Gal-lectin-based DNA
vaccine. Vaccine. 20, 3244-3253
•Houpt E., Barroso L., Lockhart L., Wright R., Cramer C., Lyerly D., Petri
W.A. (2003) Prevention of intestinal amebiasis by vaccination with
the Entamoeba histolytica Gal/GalNac lectin. Vaccine. 22, 611–617
•Kelly P, Farthing M (2005) Protozoal gastrointestinal infections
Medicine 33: 4 , 81-83.
•Stanley S.L. (2003) Amoebiasis. The lancet. 361,1025-1034 (review)