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1. cvcv Introduction cvcv Disease biology cvcv Epidemiology cvcv Clinical cvcv Summary Amoebiasis E-Learning Module Click here to begin Matt Pugh

2. cvcv Introduction cvcv Disease biology cvcv Epidemiology cvcv Clinical cvcv Summary 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.

3. cvcv Introduction cvcv Disease biology cvcv Epidemiology cvcv Clinical cvcv Summary • 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.

4. cvcv Introduction cvcv Disease biology cvcv Epidemiology cvcv Clinical cvcv Summary 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.

5. cvcv Introduction cvcv Disease biology cvcv Epidemiology cvcv Clinical cvcv Summary Causative Organism • The causitive orgainism is parasitic protazoan, called Entamoeba histolytica. • 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). • This has affected our understanding of amoeba distribution. Many suspected cases of E. histolytica carrier, may simply have been E. dispar colonisation • The WHO recommendes that E. histolytica colonisation should be treated, however, treatment is unnecessary for E. dispar colonisation Causative Organism Life Cycle and transmission 1 Life Cycle and transmission 2 Pathogenesis 1 Pathogenesis 2 Self Assessment E. Histolytica

6. cvcv Introduction cvcv Disease biology cvcv Epidemiology cvcv Clinical cvcv Summary Mouth - Cyst ingested Excyst to trophozoite Passed in stool Amoebic disease Cyst Trophozoite Invades gut mucosa – cyst formation Life cycle and transmission 1 • Entammoeba histolytica has a biphasic life cycle, existing in two forms; as an infectious cyst and an amoeboid trophozoite Causative Organism Life Cycle and transmission 1 Life Cycle and transmission 2 Pathogenesis 1 Pathogenesis 2 Self Assessment

7. cvcv Introduction cvcv Disease biology cvcv Epidemiology cvcv Clinical cvcv Summary Life cycle and transmission 2 • 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. Causative Organism Life Cycle and transmission 1 Life Cycle and transmission 2 Pathogenesis 1 Pathogenesis 2 Self Assessment

8. cvcv Introduction cvcv Disease biology cvcv Epidemiology cvcv Clinical cvcv Summary Pathogenesis 1 • 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. Causative Organism Life Cycle and transmission 1 Life Cycle and transmission 2 Pathogenesis 1 Pathogenesis 2 Self Assessment

9. cvcv Introduction cvcv Disease biology cvcv Epidemiology cvcv Clinical cvcv Summary Pathogenesis 2 • Hepatic Pathology • Trophozoites invading the colonic mucosa may enter the hepatic circulation and reach the liver Causative Organism • Well circumscribed abscesses are formed in the liver containing liquefied cells surrounded by inflammatory cells and trophozoites • Adjacent parenchyma is usually unaffected Life Cycle and transmission 1 Life Cycle and transmission 2 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

10. cvcv Introduction cvcv Disease biology cvcv Epidemiology cvcv Clinical cvcv Summary Questions • 1) Which of the following organisms is the pathogenic causitive agent of amoebiasis, and which is a commensal? • Entamoeba histolytica …………………………………………. • Entamoeba dispar ………………………………………………. • Draw a simple diagram oulining the life cycle of entamoeba histolytica. • Which two organs does E. histolytica primarily invade? • What is the name and mechanism of action of the peptide responsible for cell lysis and apoptosis in the mucosa? • What is the name of the enzyme group released by trophozoites to digest the extra-cellular matrix Causative Organism Life Cycle and transmission 1 Life Cycle and transmission 2 Pathogenesis 1 Pathogenesis 2 Self Assessment Reveal Answers

11. cvcv Introduction cvcv Disease biology cvcv Epidemiology cvcv Clinical cvcv Summary Mouth - Cyst ingested Excyst to trophozoite Passed in stool Amoebic disease Cyst Trophozoite Invades gut mucosa – cyst formation Answers • 1) Which of the following organisms is the pathogenic causitive agent of amoebiasis, and which is a commensal? • Entamoeba histolytica ……………Pathogen……………. • Entamoeba dispar ……………Commensal………………. • Draw a simple diagram oulining the life cycle of entamoeba histolytica. • 3) Which two organs does E. histolytica primarily invade? • Colon and liver • 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 extra-cellular matrix ? • Cysteine proteases Causative Organism Life Cycle and transmission 1 Life Cycle and transmission 2 Pathogenesis 1 Pathogenesis 2 Self Assessment

12. cvcv Introduction cvcv Disease biology cvcv Epidemiology cvcv Clinical cvcv Summary Epidemiology • 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. Epidemiology Susceptibility Self Assessment 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

13. cvcv Introduction cvcv Disease biology cvcv Epidemiology cvcv Clinical cvcv Summary Susceptibility • Generally considered to affect children and adults, of both sexes equally. However, some data and anecdotal evidence suggests a male predominance. • Amoebic liver abscesses are most common in males, 18-55. • 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. Epidemiology Susceptibility Self assessment

14. cvcv Introduction cvcv Disease biology cvcv Epidemiology cvcv Clinical cvcv Summary Questions • How many deaths are caused by amoebiasis each year? • 1000 – 5000 b) 40,000-100,000 c) 500,000-1,000,000 • Which part of the world is amoebiasis primarily found? • Developed countries b) Tropical and subtropical c)Cold climates • Does amoebiasis affect males or females more? • Apart from poor sanitation, what other risk factor pre-dispose to amoebiasis infection? Epidemiology Susceptibility Self assessment Reveal Answers

15. cvcv Introduction cvcv Disease biology cvcv Epidemiology cvcv Clinical cvcv Summary Answer • How many deaths are caused by amoebiasis each year? • 1000 – 5000 b) 40,000-100,000 c) 500,000-1,000,000 • Which part of the world is amoebiasis primarily found? • Developed countries b) Tropical and subtropical c)Cold climates • 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. Epidemiology Susceptibility Self assessment

16. cvcv Introduction cvcv Disease biology cvcv Epidemiology cvcv Clinical cvcv Summary Presentation • 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 Presentation Diagnosis Treatment and Management Vaccine Development 1 Vaccine Development 2 Vaccine Development 3 Self Assessment

17. cvcv Introduction cvcv Disease biology cvcv Epidemiology cvcv Clinical cvcv Summary Diagnosis • 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 Presentation Diagnosis Treatment and Management Vaccine Development 1 Vaccine Development 2 Vaccine Development 3 Self Assessment

18. cvcv Introduction cvcv Disease biology cvcv Epidemiology cvcv Clinical cvcv Summary Treatment and Management • 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 • Boiling water for at least ten minutes kills amoebic cysts effectively. Chlorine and iodine tablets are not thought to be 100% effective. Presentation Diagnosis Treatment and Management Vaccine Development 1 Vaccine Development 2 Vaccine Development 3 Self Assessment

19. cvcv Introduction cvcv Disease biology cvcv Epidemiology cvcv Clinical cvcv Summary Vaccine Development 1 • 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 Presentation Diagnosis Treatment and Management Vaccine Development 1 Vaccine Development 2 Vaccine Development 3 Self Assessment

20. cvcv Introduction cvcv Disease biology cvcv Epidemiology cvcv Clinical cvcv Summary Macrophage IgA, MAb, antiserum Key Activate Attack Inhibit Mucosa epithelial cell Gal-lectin NO Cytokines e.g INF-γ Trophozoite Th1 cell 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 T-cells 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. Presentation Diagnosis Treatment and Management Vaccine Development 1 Vaccine Development 2 Vaccine Development 3 Self Assessment

21. cvcv Introduction cvcv Disease biology cvcv Epidemiology cvcv Clinical cvcv Summary Vaccine Development 3 • Both Gal-lectin classical and DNA based vaccines have been tested in murine models. • 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. Protection conferred from purified and recombinant vaccines Presentation Gal-lectin classical vaccine Purified (lectin) and recombinant gal-letin (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. Diagnosis Protection % Treatment and Management Vaccine Development 1 Vaccine Development 2 Production of DNA gal-lectin DNA vaccine in murine model. Vaccine Development 3 170 35 Self Assessment Gene coding for portion of heavy gal-lec subunit isolated Transfected into plasmid Plasmid injected intra-muscularly Muscle cells take up and incorporate gal-lec sequence in DNA Protein expressed and immune response induced

22. cvcv Introduction cvcv Disease biology cvcv Epidemiology cvcv Clinical cvcv Summary Questions • What are the symptoms of gastrointestinal amoebiasis? • What are the symptoms of hepatic amoebiasis? • Why is a good travel history important in diagnosis of amoebiasis? • What investigations can be performed to confirm a diagnosis? • Name two drugs and dosage regimes that can be used to treat amoebiasis. • 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? Presentation Diagnosis Treatment and Management Vaccine Development 1 Vaccine Development 2 Vaccine Development 3 Reveal Answer Self Assessment

23. cvcv Introduction cvcv Disease biology cvcv Epidemiology cvcv Clinical cvcv Summary Answers • 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. • 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) 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. • 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. • 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 • Does Gal-lectin induce a Th1 or Th2 cell mediated immune response? • Th1 cell mediated response Presentation Diagnosis Treatment and Management Vaccine Development 1 Vaccine Development 2 Vaccine Development 3 Self Assessment

24. cvcv Introduction cvcv Disease biology cvcv Epidemiology cvcv Clinical cvcv Summary 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 Summary References

25. cvcv Introduction cvcv Disease biology cvcv Epidemiology cvcv Clinical cvcv Summary References and further reading • Gaucher D., Chadee K. (2003). Prospect for an Entamoeba histolytica Gal-lectin-based vaccine. Parasite Immunology. 25, 55–58 (review) • 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) Summary References