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Para Lect Outline Chapter 2

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   1 Parasitology Lecture Outline: Chapter 2 Intestinal Amoeba    Seven species of amoeba occur in Humans, includes: -   Pathogenic: Entamoeba histolytica  -   Commensals: Entamoeba dispar  , Entamoeba moshkovskii  , Entamoeba hartmanni  , Entamoeba coli  , Endolimax nana , and Iodamoeba butschlii       Entamoeba polecki   - an intestinal ameba of pigs and monkeys but it has been occasionally detected in humans. It can be the probable cause of diarrhea    Binary fission: Trophozoites       Nuclear division then divide again after excystation: Cyst-forming amebae ( most )   Entamoeba histolytica      Subphylum: Sarcodina  Superclass: Rhizopodia      Class: Lobosea  Order: Amoebida      Family: Entamoebidae  Genus: Entamoeba      Genus characteristic: -   Vesicular nucleus  - a centrally located small karyosome.   -   Varying numbers of chromatin granules adhering to the nuclear membrane.    Can distinguish species of  Entamoeba  EXCEPT: Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii.      Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii - are morphologically identical and of the same size. -   PCR , RFLP and typing with monoclonal antibodies    –   differentiated the 3.    E. hartmanni     –     before referred to as “small race” of E. histolytica now are differentiated by size.    Parasite Biology : -   Psedopod-forming non-flagillated protozoan  parasite. -   Most invasive  in all Entamoeba parasites -   Only member that causes: colitis and liver abscess  -   Life cycle: Infective cyst and invasive trophozoite form.  -   Hosts: Human and primates . -   Quadrinucleate cyst  - resistant to gastric acidity and desiccation, and can survive in a moist environment for several weeks -   Mode of transmission: Ingestion of cyst from fecally-contaminated material and -   Other modes of transmission: Venereal transmission through fecal-oral contact or or direct colonic inoculation through contaminated enema equipment. -   Excystation  - where a cyst undergoes nuclear followed by cytoplasmic division to form eight trophozoites -   E. histolytica trophozoites : highly motile and  possess pseudopodia (locomotory structure). Multiply by binary fission Movement: directional and progressive.  -   Hyaline pseudopodium  -   formed when the clear, glasslike ectoplasm, or outer layer is extruded, and the granular endoplasm flows into it. -   Ingested RBC:  pale, greenish, refractile bodies in the cytoplasm of the ameba .  -   Cyst form (spherical) :      Reflactile hyaline wall    # of nuclei: 1-4    Chromotoidal bars appearance: Rod-shaped or Cigar-shaped    Pathogenesis and Clinical Manifestations -   Mechanism for virulence : Enzyme production, contact-dependent cell killing, and cytophagocytosis. -   HLA complex: specific allele that associate to  Entamoeba histolytica  -    Entamoeba histolytica  adhere to the colonic mucosa through Gal Lectin  -   Difference of  E. histolytica  and  E. dispar  : PCR, enzyme-linked immunosorbent assay (ELISA), and  isoenzyme analysis.    E. dispar   is more prevalent  than  E. histolytica  but  E. histolytica  in most endemic communities are asymptomatic . -   Amebic colitis  - gradual onset of abdominal pain and diarrhea with or without blood and mucus in the stools. Fever only 1/3 third of the patient experience when having amebic colitis. Most serious complications: perforation and secondary bacterial peritonitis. Less common complications: Fever and significantly elevated leukocyte count.  -   Children: Fulminant colitis    –   with severe bloody diarrhea, fever, and abdominal pain. -   Ameboma  - a mass-like lesion with abdominal  pain and a history of dysentery. It can be mistaken for carcinoma. -   Amebic liver abscess (ALA)    –   most common extra-intestinal form of amebiasis. Cardinal Manifestation: Fever  and right upper quadrant pain. Most serious complications: Pericardial rupture  (70% mortality rate), Pleural puncture  (15-30% mortality rate) and superinfection . 2 nd  most serious complication: Intraperitonial rupture  (2 to 7.5% of cases). Differential diagnoses: Pyogenic liver abscess, tuberculosis of the liver, and hepatic carcinoma -   Secondary amebic meningoencephalitis  - considered in cases of amebiasis with abnormal mental status. -   Activated T-cells kill  E. histolytica  by:   2    Directly lysing trophozoites in a contact-dependent process.    Producing cytokines which activate macrophages and other effector cells such as neutrophils and eosinophils.    Providing helper effect for B-cell antibody  production. -   Interferon (IFN) and interleukin (IL-2)    –   have a role in activating macrophages for amebicidal activity -   Macrophages    –   produce nitric oxide which is lethal to trophozoites. Nitric oxide production stimulator: Tumor necrosis factor -   States that favors amebic survival: T-cell hyporesponsiveness, suppressed proliferation and cytokine production, depressed delayed-type hypersensitivity (DTH), and macrophage suppression. -   Acute amebic colitis should be differentiated from bacillary dysentery’s etiology:  Shigella ,  Salmonella , Campylobacter  , Yersinia , and enteroinvasive Escherichia coli -   Genital amebiasis should be differentiated from: carcinoma, tuberculosis, chancroid , and lymphogranuloma venereum    Diagnosis -   Microscopic detection of trophozoite and cyst in stool specimen - Standard method of  parasitologic diagnosis. -   Trophozoite detection  - fresh stool specimens should be examined within 30 minutes from defecation. -   DFS with saline solution  - can observe trophozoite motility. -   Saline and methylene blue    –   stains  Entamoeba  species  to blue to differentiate them from white  blood cells. -   Saline and iodine  –    nucleus and karyosome can  be observed to differentiate  E. histolytica from the non-pathogenic amebae. -   Formalin Ether/Ethyl Acetate Concentration Test (FECT) and Merthiolate Iodine Formalin   Concentration Test (MIFC)    –   more sensitive than DFS for cyst detection. -   Morphological structures to differentiate  E. histolytica  from non-pathogenic species: size of the cyst, number of nuclei, location and appearance of the karyosome, the characteristic appearance of chromatoid bodies, and presence of cytoplasmic structures such as glycogen vacuole. -   Robinson’s and Inoki medium for stool culture  - more sensitive than stool microscopy, but is not routinely available. -   Methods to differentiate  E. histolytica and E. dispar: PCR, enzyme-linked immunosorbent assay (ELISA), and isoenzyme analysis -   Detection of antibody in the serum - the key in the diagnosis of ALA -   Serological tests for amebic disease: Indirect hemagglutination (IHAT), counter immunoelectrophoresis (CIE), agar gel diffusion (AGD), indirect fluorescent antibody test (IFAT), and ELISA  -   Indirect hemagglutination (IHAT)    –   determination of antibodies of past infections even as long as 10 years ago. -   ELISA, AGD, and CIE - determination of antibodies of past infections for short durations that last for a few months. -   Ultrasound, CT scan, and MRI  - non-invasive and sensitive methods in early detection of ALA -   Objective for amebiasis treatment: Curation of invasive disease at both intestinal and extraintestinal sites and elimination of the passage of cysts from the intestinal lumen . -   Metronidazole  - drug of choice for the treatment of invasive amebiasis    Other drugs: tinidazole and secnidazole -   Diloxanide furoate  - drug of choice for asymptomatic cyst passers -   Percutaneous drainage of liver abscess    –   indication for patients who do not respond to metronidazole, who need prompt symptomatic relief of severe pain and left lobe abscess.    Epidemiology -   E. histolytica    –   believed to infect 500 million people , or 10%   of the world’s population. But recent studies said that the true prevalence of amebiasis is 1% - 5% worldwide , 50 million  are infected and 40,000  –   100,000 deaths  due to amebiasis in a year worldwide -   Amebiasis  - third most important parasitic disease, after malaria and schistosomiasis, and second to malaria as the top cause of mortality among parasitic protozoans -   Major reservoir of E. histolytica infection: Humans   -   Means of infection: Ingestion of food and water with cyst and direct fecal- oral contact .   -   Amebic infection  - prevalent in the Indian subcontinent, Africa, East Asia, and South and Central America -   Groups that  E. dispar   is prevalent: immigrants, travelers from endemic countries, homosexual males (men having sex with men), HIV patients, and institutionalized people.      Prevention and Control -   Proper use of latrines -   Proper hygiene  –   washing of hands   3 -   Drinking water should be boiled or filtered -   Avoidance of the usage of night soil for fertilizer. -   Vegetable that are eaten raw should be washed thoroughly. -   Food handlers should be screened for cyst carriage -   Asymptomatic cyst carriers should be treated. -   Vaccines  - can be a cost-effective and potent strategy for amebiasis prevention and eradication Commensal Amebae    Reasons of commensal amebae is present in stools: Amebae may be mistaken for the pathogenic Entamoeba histolytica  and an indication of fecal contamination of food or water .    Parasite Biology : -   Genus Entamoeba   - spherical nucleus with nuclear membrane lined with chromatin granules and a small central karyosome. -   Trophozoite  –   usually 1 nucleus. -   Genus Endolimax     –   has a vesicular nucleus with large and irregularly-shaped karyosome anchored to the nucleus by achromatic fibrils. -   Genus Iodamoeba   - has large, chromatin-rich karyosome surrounded achromatic globules and anchored to the nuclear membrane by achromatic fibrils. -   Stages: Trophozoite, precyst, cyst, and metacystic trophozoite except Entamoeba  gingivalis.      Entamoeba gingivalis - no cyst stage, and does not inhabit the intestines -   Mode of transmission by commensal intestinal amebae: Ingestion of viable cyst in food or water by humans. -   Excystation  - occurs in the alkaline environment of the lower small intestines -   Metacystic trophozoites    –   colonize large intestine and live on the mucus coat covering the intestinal mucosa. -    Non-invasive and do not cause disease. -   Reproduction: trophozoite  –    binary fission  -   Encystation  - amebae pass through the lower colon where colonic contents are more dehydrated.    Entamoeba dispar -   Entamoeba dispar     –   morphological similar to  E. histolytica , but DNA and ribosomal RNA differs. -    Entamoeba moshkovskii    Reported area: North America, Italy, South Africa, Bangladesh, India, Iran, and Australia .    Non-pathogenic  species that the morphologically is the   same  from  E. histolytica and  E. dispar  . Only differs biochemically  and genetically .    Physiology : Osmotolerant - able to grow at room temperature, able to survive at temperatures ranging from 0 to 41°C.    Entamoeba hartmanni    -   Appearance: relatively similar to E. histolytica  but smaller size . -   Trophozoites : diameter is 3  to 12 μm  -   Mature cysts : 4-10 μm  in measurement, quadrinucleated ,, and rod-like chromlotoid   bars with rounded  or square   end . -   Does not ingest RBC.    Entamoeba coli    -   More common than other human amebae. -   Habitat or Location : Large intestine -   Trophozoites : diameter is 15 to 50 μm  -   Difference of  E. coli  trophozoites from  E. histolytica trophozoites: More vacuolated or granular endoplasm with bacteria and debris, no red blood cells, a narrower and less-differentiated ectoplasm, broader and blunter pseudopodia for feeding than locomotion, more sluggish and undirected movements and thicker, irregular  peripheral chromatin with a large, eccentric karyosome in the nucleus. -   Difference of E. coli cyst from  E. histolytica cyst: larger size, more nuclei, more granular cytoplasm and splinter-like chromotoidal bodies, -   Iodine staining - reveals dark-staining,  perinuclear masses, which are actually glycogen. Surrounding the nucleus, it is more characteristic of  E. coli compared to  E. histolytica .    Entamoeba polecki -   Host: Pigs  and monkeys. Habitat or Location: Intestine  -   Rare host: Humans . Trophozoite motility: Sluggish  -   Karyosome location: Central   -   Appearance of cyst: Uninucleated  and angular  or pointed   chromatoidal   bars      Entamoeba chattoni    -   Host: Apes  and monkeys  -   Morphologically identical  to  E. polecki  -   Identification of  E. chattoni : isoenzyme analysis    Entamoeba    gingivalis   -   Host: Humans -   Habitat or Location : Mouth or oral cavity  specifically in the surface of the gum and teeth , in gum sockets  and sometimes in the tonsillar crypts.  -   Trophozoites: 10 to 20 μm  -   Locomotion: Moves quickly  and has numerous blunt pseudopodia .   -   Food vacuoles: Contain mostly leukocytes  -   No cyst stage   4 -   Mode of transmission : Kissing, droplet spray and  by sharing utensils.    Endolimax    nana   -   Habitat or Location : Large intestine. -   Occurs same frequency as E. coli    -   Small trophozoite: 5 to 12 μm  in diameter and have a sluggish movement .   -   Locomotory structure : Blunt, hyaline pseudopodia. -   Karyosome : large and irregular karyosome -   Food vacuoles: Contain bacteria -   Cyst: 5 to 12 μm and  quadrinucleated.    Iodamoeba   bütschlii    -   Trophozoite: Average 9  –   14 μm in diameter    -    Nucleus : Large and  vesicular nucleus -   Karyosome: Large  and central karyosome surrounded by  achromatic granules  -    No peripheral chromatin -   Cyst : about 9 to 10 μm in diameter, uninucleated and has a large glycogen body that stains dark  brown with iodine    Diagnosis -   Stool examination -   FECT and iodine stain    –   useful to differentiate the species -   For  E. gingivalis - swab between the gums and  teeth for trophozoites examination   -   Formed stool: Cyst recovered   -   Watery or semi-formed stool: Trophozoite recovered   -   DFS  –     best for trophozoite recovering.   -   FECT  and zinc sulfate flotation    –   best for cyst recovering    Treatment    –   no treatment needed because they are non-pathogenic    Prevention and Control -   Contraction of the organism may be prevented through proper disposal of human waste and good  personal hygiene. Free-living Pathogenic Amebae Acanthamoeba spp.    Parasite biology -   An ubiquitous, free-living ameba that is the etiologic agent of  Acanthamoeba keratitis and granulomatous amebic encephalitis. -   Has 2 stages : Trophozoite stage:    “thorn - like”  acanthopodia (Appendages)     Nucleus: single  and centrally  located     Nucleolus: Densely stained. Endosome: Large      Cytoplasm: Fine  and granulated. Contractile vacuoles:  Large      Locomotory structure : Small and  spiny filament acanthapodia      Feeding: Gram- negative bacteria, blue-green algae , or yeasts .    Feeding adaptation: corneal epithelial   cells  and neurologic tissue  via phagocytosis and lytic enzymes secretion.    Reproduction: Asexual  or binary fission  for motile trophozoite.    Replication: Mitosis    Infective stage      Can enter through: Eye, the nasal passages to the lower respiratory tract, ulcerated or  broken skin -   Cyst stage:  highly dependent cyst that transform when environmental condition is not favorable. -   Aquatic organism found in a myriad of natural and artificial environments, and even in contact lens cleaning solutions -   Presence of naturally-occurring bacterial endosymbionts -   Can serve as a reservoir host for: Legionella spp. , mycobacteria , and gram-negative bacilli  such as E. coli  .    Pathogenesis and Clinical Manifestation -    Acanthamoeba Keratitis      Acanthamoeba  - first described as an opportunistic ocular surface pathogen causing keratitis.    Associated with the use of improperly disinfected soft contact lenses, particularly those which are rinsed with tap water or contaminated lens solution.    Can lead to disease in the lungs and brain (GAE)    Symptoms: severe ocular pain, blurring of vision and can even be Corneal ulceration with progressive corneal infiltration    Primary amebic infection or secondary  bacterial infection may lead to hypopyon formation.    Progression of infection may cause scleritis and iritis, and may even lead to vision loss. -   Granulomatous Amebic Encephalitis    Usually occurs in immunocompromised hosts and those undergo chemotherapy and anti-rejection medications.    AIDS epidemic in 1980’s increased  the numbers  of person with GAE      Signs and symptoms : Destruction of brain tissue and associated with meningeal irritation.    Systemic manifestations : Fever, malaise, and anorexia    Neurologic symptoms : Increased sleeping time, severe headache, mental status changes, epilepsy, and coma

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