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