TOPIC OUTLINE I. Amoeba Parasitic Biology a. Morphology b. Life Cycle and Reproduction c. Intestinal and Oral Amoebas II. Entamoeba hystolytica a. General Features b. Life Cycle c. Pathogenesis and Clinical Manifestations d. Diagnosis e. Treatment, Prevention, and Control III. Dentamoeba fragilis a. General Features b. Pathogenesis c. Diagnosis d. Treatment IV. Commensals a. General Features b. Entamoeba dispar c. Entamoeba hartmanii d. Entamoeba coli e. Entamoeba gingivalis f. Entamoeba polecki
o o
g. Endolimax nana AMOEBA PARASITIC BIOLOGY
Morphology Cytoplasm o Ectoplasm
Outer, thin layer for movement, ingestion,
excretion, respiration, protection o Endoplasm
Voluminous inner which contains nucleus
Nucleoplasm o Nuclear membrane enveloping a fine reticulum filled with nuclear sap and chromatin
Vesicular concentrated in a single mass
Granular diffused o Karyosome: centrally, deeply stained for promitosis Life Cycle and Reproduction Reproduction is asexual/ binary fission Trophozoite INVASIVE o Pathologic state and mature form o Vegetative, destroyed by unfavorable environment o Has the ability to colonize and/or invade the large bowel (cysts dissolve in the alkaline SI and hatches in the LI) o Present in diarrheal stool Cyst INFECTIVE o Inactive trophozoite inside a shell o Survives in any environment; resistant to gastric acidity and desiccation
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Quadrinucleated Present in formed stool
Intestinal and Oral Amoebas
Intestinal Amoebas o Have trophozoite and cyst stages except Dientamoeba fragilis (only cyst stage) o Found in the large intestine o Mode of transmission:
Oral-fecal route
Venereal transmission
Contaminated instruments (forceps)
Via intestinal roundworms (D. fragilis: thru
eggs or adults of E. vermicularis and A. lumbricoides) o Examples:
Entamoeba histolytica
Entamoeba coli
Entamoeba dispar
Endolimax nana
Iodamoeba butschii
Dientamoeba fragilis Oral Amoeba o No cyst stage; Needs direct passage for survival o Found in the oral cavity o Mode of transmission:
Kissing
Sharing of utensils, toothbrush
o Example:
Entamoeba gingivalis
ENTAMOEBA HYSTOLYTICA
General Features Most in invasive of the Entamoeba family Pseudopod-forming, non-flagellated Eukaryotic with 1-4 nuclei o undergoes nuclear division followed by cytoplasmic divisions to form 8 trophozoites Causes intestinal amebiasis, colitis and Amoebic Liver Abscess (ALA) Unusual features o Presence of nuclear-encoded mitochondrial genes such as pyridine nucleotide transhydrogenase and HSP 60 o Lack of organelles that morphologically resemble mitochondria o No rough ER and no Golgi apparatus o Tunicamycin inhibits protein glycosylation o Lack of glutathione metabolism o Use of pyrophosphate instead of ATP at several steps in glycolysis
Lecture Title o o
Inability to synthesize purine nucleotides
de novo End product of CHO metabolism are ethanol and CO2, and under aerobic conditions, acetate
Life Cycle 1. Cysts are passed in feces. 2. Infection of E. histolytica occurs by ingestion of mature cysts in focally contaminated food, water, or hands. 3. Since cyst is resistant to stomach acid, it passes to small intestine where excystation occurs, releasing trophozoites which migrate to the large intestine. 4. Trophozoite multiply by binary fission and produce cysts, both stages passed in the feces. 5. Trophozoite passed in the stool are rapidly destroyed once outside the body, and if ingested would not survive exposure to gastric environment 6. In many cases, trophozoites remain confined to the intestinal lumen (noninvasive infection) of asymptomatic carriers, passing cysts in their stool. In others, trophozoites invade intestinal mucosa (intestinal disease) or through bloodstream, extraintestinal sites such as liver, brain, and lungs (extraintestinal disease)
Fig1. Life cycle of E. hystolytica.
Pathogenesis and Clinical Manifestations 1. Sites
Cecum
Ascending colon
Hepatic flexure
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Liver o Via portal vein (Hematogenous spread) periportal inflammation and amoebic hepatitis o Via direct extension from intestinal ulcer (flask-shaped ulcers when trophozoite erode through lamina and extend laterally): Amoebic Liver Abscess
necrotic
proteinaceous debris
anchovy sauce-like aspirate
odorless (may not be contaminated)
2. Invasion due to virulence factors
Gal/ GalNac lectin
o Mediates adherence to host cells
Amebapores o Form spores on host cell membranes
Cysteine proteinases o Cytopathic for host tissues 3. Types of invasion
Direct extension liver, lungs, skin
Hematogenous liver, lungs, brain, heart,
etc. 4. Signs and Symptoms
Liver: tenderness, hepatomegaly, right
upper quadrant pain
Blood: fever
Lungs: dyspnea, hemoptysis
Intestine: diarrhea, hyperperistalsis,
ulcerations
Brain: headache, convulsion, paralysis
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5. Signs and Symptoms of Complications
Asymptomatic o Majority of the cases o Cyst carrier state (passed out in stools) o In endemic communities
Amebic colitis o Most common and serious form of disease o Intestinal perforation o Secondary bacterial peritonitis o Occurs in 60% fulminant colitis o Gradual onset of the abdominal pain o Diarrhea with/without blood o Mucus in the stool o Fever occur in 1/3 of patients o Diarrhea alternating with constipation o Children: fulminant colitis with severe bloody diarrhea, fever and abdominal pain
Ameboma o Occur in less than 1% of the cases o Mass-like lesion with abdominal pain o History of dysentery o Mistaken for carcinoma o May also be asymptomatic
Amebic Liver Abscess (ALA)
o Most common form of extra-intestinal amebiasis o Rupture into the pericardium (most serious; 70% mortality rate), pleura,
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superinfection, intraperitoneal rupture
(second most common) o Only 30% with concurrent diarrhea o Invasive process of trophozoite o Cardinal manifestations
Acute cases (<2weeks): fever, right
upper quadrant pain
Pain: local and referred to the right
shoulder
Liver: tender (esp. in acute cases)
Hepatomegaly (50% of cases)
Chronic (>2 weeks): wasting disease
with weight loss rather than fever Secondary Amebic Meningoencephalitis o Occurs in 1-2% ; Considered in cases of amebiasis with abnormal mental status Renal involvement (rare) o Caused by extension of ALA or retroperitoneal colonic perforation Genital involvement o Caused by fistulae from ALA and colitis o Primary infection through sexual transmission
Diagnosis 1. Microscopic Examination (Standard)
3 consecutive stool examinations in 3 days
Detection of trophozoite in fresh stool 30
min after defecation
Highly motile trophozoites with
unidirectional movement
Detection of E. histolytica trophozoites with
ingested RBCs is diagnostic of amoebiasis 2. Direct Fecal Smear
NSS
Saline and methylene blue stain
Entamoeba species stains blue
differentiating them from WBC
NSS and iodine: nucleus and karyosome
obserbed to differentiate pathogenic from nonpathogenic species 3. Concentration Techniques
More sensitive than DFS for detection of
cysts
Formalin Ether Concentration Test
Merthiolate Iodine Formaline Concentration
Test 4. Stool Culture
Robinsons and Inokis Medium more
sensitive than microscopy but is not routinely available 5. PCR
Expensive
Lecture Title
Helps differentiate one species from another
(E. histolytica vs E. dispar) 6. ELISA
80-90% accuracy
Assays for Amebic Gal/GalNac lectin
antigens 7. Liver Ultrasound
Trophozoites surround liver abscesses
Right upper lobe of the liver most infected
8. Serology
Indirect hemagglutination
Counter immunoelectrophoresis
Agar gel diffusion
Indirect fluorescent antibody test
Enzyme-linked immunosorbent assay
9. Colonoscopy
Done in ascending colon and cecum
Wet preparations from aspirations and
scrapings from ulcers 10. Radiographic studies
CT scan, MRI, and ultrasound
11. Aspiration of the tissue
Periphery (ALA)
Treatment, Prevention, and Control
Percutaneous drainage of abscess for
nonresponders Environmental sanitation Health education and promotion Boiling of drinking water for 20mins Avoidance of night soil Prompt diagnosis and treatment Vaccine (experimental)- serine rich E. hystolitica protein (SREAP), adherence lectins (GAL/GALNAC lectins), 29kDa cysteine rich amoebic antigen
DENTAMOEBA FRAGILIS
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Host Defense Mechanism
1. Nonspecific Defense o First line mucin o Second line complement system 2. Specific Defense o Third line cellular mediated immunity
Against trophozoite
Activated T cells kill E.
histolytica
direct lysis
cytokines
helper effect from antibody
prod. Drug of Choice (DOC) - Metronidazole Cyst removal through filtration and boiling (not chlorination)
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General Features Flagellated with only trophozoite stage known Rosette nucleus, nuclear membrane has no chromatin Binucleated, resembles Trichomonas Infrequently found in association with other parasitic intestinal pathogens with the exception of pinworm infestations, where the prevalence of infections with both organisms is ninefold greater than would be expected based on just a random association Also found in Ascaris and Enterobius Neither its epidemiology nor its route of transmission is completely known Mode of Transmission: o Believed to be transmitted between human hosts inside helminth eggs or larvae, particularly those of Enterobius vermicularis o Evidence: frequent association between the two organisms and the presence of bodies inside the helminth eggs, which resemble D. fragilis Pathogenesis Irritation of mucosal lining with excretion of excess mucus and hypermotility of bowel Colicky abdominal pain and abdominal tenderness Bloating sensation and flatulence Intermittent diarrhea with excess mucus Anal pruritus (E. vermicularis) Non-invasive: no bleeding Mistaken for irritable bowel syndrome (IBS) Diagnosis Microscopic stool exam (same as in E. histolytica )
Lecture Title
Treatment Drug of Choice (DOC) - Iodoquinol for 20 days Treat also E. vermicularis Proper sanitation Alternative Treatments o Tetracycline o Metronidazole
COMMENSALS
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General Features No need for treatment, just indicative of food contamination Entamoeba dispar Non-invasive protozoan Morphologically similar to E. histolytica In controlled in numbers or if immunocompromised can cause colitis Commonly found in captive primates Live and multiply in intestinal crypts of the large intestine mucosa and feed on starches and mucus secretions Entamoeba hartmanii Similar to E. histolytica but much smaller Does not ingest RBC More sluggish in movement Mature cysts measures from 5-10 um, quadrinucleate and have coarse cytoplasm Immature cysts have chromatoidal bars which may be short with tapered ends or thin and bar-like Entamoeba coli Largest intestinal amoeba Survives putrefaction Feed on bacteria, other protozoa, yeasts and blood cells Endosomes eccentrically arranged Cysts has chromatoidal bars that have splintered ends May have as much as 8-16 nuclei Used to determine whether water sources are contaminated with fecal wastes Entamoeba gingivalis Only found in mouth First amoeba in humans to be described Only found in trophozoite form and do not form cysts Move quickly because of blunt pseudopodia
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Central endosome Chromatin concentrated in the inner surface of inner surface Food vacuoles contain cellular debris, bacteria and blood cells Lives in the surface of teeth and gums even at crypt of tonsils Entamoeba polecki Usual parasites of pig and monkeys rarely in humans Nonpathogenic in humans Have on nucleus Cysts have one nucleus but may reach binucleate stage Endolimax nana Smallest amoebas Live both in vertebrates and invertebrates Vesicular nucleus Endosome comparatively large and irregular Endosome attached to the nuclear membrane via achromatic threads Lives in the human large intestine near the cecum Feeds on the bacteria Pseudopodia are short and blunt Food vacuoles contain bacteria, plant cells and debris Undergoes encystment
Fig2. Morphology of trophozoite and cyst stage of
E. hystolytica, E. hartmanii, E. coli, E. polecki, Endolimax nana, Iodamoeba butschlii.
