16/07/64 Objectives • be able to identify and describe the Pathogenic Amoebae pathogenic amoebae in terms of morphology, life cycle, pathology, symptoms, transmission and diagnosis Human Pathogen II Assoc. Prof. Dr. Anchalee Wannasan 1 Entamoeba histolytica • 2 stages in the life cycle • Disease: amoebiasis • Distribution: worldwide - second leading cause of death after malaria - developing countries: poor sanitation areas - developed countries : immigrants or travelers from endemic areas • Transmission: Cyst (dormant form) Trophozoite (active form) - fecal-oral route - venereal transmission (homosexuals) • Infective satge: mature cyst with 4 nuclei 1 16/07/64 Cyst ~10-20 µm • round or oval shape • thin and tough cyst wall • contains 1, 2 or 4 nuclei www.medical-labs.net • centric karyosome • peripheral chromatin karyosome • chromatoid bar with rounded ends (Cigar-like) Chromatoid body Peripheral chromatin www.medical-labs.net 5 7 2 16/07/64 Trophozoite ~15-60 µm • irregular shape • a round nucleus • small, centric karyosome • peripheral chromatin scattered evenly throughout the inner nuclear membrane • fine granular endoplasm (food vacuoles + RBC) 10 Rapid movement by finger-like pseudopodia 12 3 16/07/64 Pathology and symptoms 1. Intestinal amoebiasis (Invasive amoebiasis) • Amoebiasis is a disease caused by E. histolytica. incubation period is variable, (mostly 1-4 w) • Forms of amoebiasis characterized by 4 markers: 1) Intestinal amoebiasis o amoebic dysentery (mucus and bloody diarrhea) - 90% is asymptomatic cyst passers (E. dispar ?) o trophozoites with ingested RBC (feces, rectum biopsy) - 10% is symptomatic (Invasive amoebiasis) o amoebic colitis (endoscopy) 2) Extraintestinal amoebiasis o positive serological test for specific Ab Ameboma Other clinical manifestations o increasing diarrhea progressing to mucous-bloody o lower abdominal pain, tenesmus o weight loss, fever o similar to other colonic diseases o WBC are rarely seen in stools o progressive involvement - Fulminant necrotitizing colitis, toxic megacolon - Acute rectocolitis - Amoebic appendititis https://www.wjgnet.com/1007-9327/full/v22/i36/8234.htm - Amoeboma (immune response to trophozoites) • Granulation tissue and fibrotic thickening caused by 15 host immune response to trophozoites, Fig A, B) 16 4 16/07/64 Amebic colitis Intestinal amebic ulcers https://cmr.asm.org/content/13/2/318/figures-only https://link.springer.com/chapter/10.1007/978-4-431-55200-0_23 1) Nodular lesion (0.1- to 0.5-cm in diameter) http://intranet.tdmu.edu.ua/data/kafedra/internal/med_biologia/classes_stud/en/med/prophylactic%20medicine/ptn/medical%20biology/1%20course/theme%2008.htm o rounded and small ulcer with slightly elevated rims • Severe dysentery with multiple ulcers in the large o necrotic center - appears dimpled or hemorrhagic bowel, and a bloody diarrhea - filled with yellowish mucous 17 18 2) Irregular lesion (1- 5 cm in length) o shallow but broad Flask-shape amoebic ulcer with elevated rims o filled with fibrin and yellow exudates 4.bp.blogspot.com https://www.researchsquare.com/article/rs-29990/v1 Irregular cecum 19 20 lesions 5 16/07/64 2. Extraintestinal amoebiasis Pathogenesis • Hematogenously spread to liver and other • glandular hyperplasia organs (lung, pericardium, brain, skin, and • stromal edema genitourinary tract, etc.) • atrophy of villi & epithelium • Liver is the most common involvement site • infiltration of neutrophils • 3–9 % of all intestinal amebiasis cases develop • deep into submucosa to amoebic liver abscess (ALA) • 2nd bacterial infection 21 (flask-shape amoebic ulcer) Amoebic liver abcess (ALA) Amoebic liver abcess .capillary obstruction in portal system (necrosis) d_Pathogenesis/figures?lo=1 https://www.researchgate.net/publication/221929027_Amoebiasis_in_the_Tropics_Epidemiology_an . pin head-like lesion, then enlarged and became abscess acute - right upper quadrant abdominal pain - fever - liver tenderness and hepatomegaly chronic - weight loss, vague abdominal symptoms https://en.wikipedia.org/wiki/Amoebic_liver_abscess Rupture of abscesses into the abdomen or chest may https://www.sciencephoto.com/media/250192/view/liver-abscess lead to death. 6 16/07/64 Diagnosis • Extraintestinal amebiasis Intestinal amebiasis • stool exam may not be helpful • Microscopic examination (stool or rectal smear) • aspirates from abscess may not find amoebae - dysenteric stool : trophozoites • biopsy from liver abscess rims (recommended) - nondysenteric stool : cysts • - differential diagnosis from E. dispar imaging procedure (ultrasound/ CT / MRI) using isozyme analysis, serological methods, PCR • serological methods (ELISA) • Biopsy from colonoscopy / sigmoidoscopy - discriminate ALA from pyogenic liver abscess - negative in Entamoeba dispar infected patient • PCR 25 26 Treatment drugs of choice : Metronidazole - effective to both intestinal and tissue amoebiasis Nitroimidazole derivatives such as secnidazole, http://medicinembbs.blogspot.com/2013/01/amoebic-liver-abscess.html nimorazole and tinidazole ALA - Percutaneous drainage or surgical aspiration Charateristics of pus from ALA: E. dispar infection does not require the treatment anchovy paste-liked color without smell 28 7 16/07/64 Pathogenic free-living amebae Common features (pathogenic FLA) Naegleria fowleri o Primary amoebic meningoencephalitis (PAM) • facultative free-living parasites Acanthamoeba spp. • generally found in the o Granulomatous amoebic encephalitis (GAE) environment o Acanthamoebic keratitis (AK) • mortality rate approach 100% Balamuthia mandrillaris o GAE • no specific therapeutic agents Sappinia pedata o GAE 29 30 Naegleria fowleri Morphology Cyst ~ 8-20 µm • known as the brain-eating amoeba • Distribution : worldwide (water and soil resources) • Disease : primary amebic meningoencephalitis most cases were immunocompetent host • Most clinical isolates are thermophilic (> 45°C). https://www.si.mahidol.ac.th/th/department/parasitology/articledetail.asp?ac_id=7&pageno=1 • 3 stages in LC: cyst , trophozoite • round with double cyst walls and flagellate (Amoeboflagellate) • big karyosome surrounded by halo • never found cyst stage in host tissue 31 8 16/07/64 Trophozoite Flagellate Life cycle ~ 10-35 µm ~ 10-16 µm reversible (Lobopodia) (flagellum) • Enflagellation test: In distilled water or nutrients depriving condition, trophozoite transforms into flagellate (2 flagella) 33 34 Risk activities: • Diving or swimming in fresh water resources • Performing ‘neti’ (nasal rinse) https://www.insider.com/neti-pot-tap-water-is-it-dangerous-brain-eating-amoeba-2018-12 • a ritual cleansing that includes nasal passages 36 9 16/07/64 Clinical manifestation Diagnosis Incubation period: 3-7 days immunocompetent host (mainly children & young adult) history of water exposure (exp. swimming) acute common cold-like symptoms: CSF sedimentation : found only trophozoite fever, rhinitis, stiff neck, sore throat and severe headache o culture on non-nutrient agar with gram (-ve) bacteria similar to that of acute bacterial and viral meningitis o enflagellation test, PCR Rapidly progression with high fever, vomiting, seizure, • brain tissues: found only trophozoite edema of lung and brain, respiratory failure, coma o staining o IFA, Immunohistochemistry Death within 10 days o PCR 37 Treatment Prevention no specific therapeutic agents Chlorine in swimming pool 1-2 ppm Combination of available drugs which can cross blood- Avoid swimming in fresh natural resources brain barrier Wear the nose clip during swimming Miltefosine or combination anti-microbial therapy: Blow the nose using sterile reagent amphotericin B, Azithromycin, rifampin, and azole drugs Intensive supportive care is required Only a few from hundred cases survive 39 40 10 16/07/64 Morphology Acanthamoeba A) Cyst ~ 5-25 µm • Free-living protozoan (facultative) parasites - uninucleated - worldwide distribution : soil, air, dust, fresh and sea water, - thick-wall polygonal , stellate, Jacucci tubs, dental irrigation unit, contact lens, etc. oval or spherical endocyst - usually concentrate at the bottom of the lake with ostioles - wrinkle ectocyst • Pathogenic to man (thermophilic) ~ 10-45 µm B) Trophozoite • Exp. A. castellani, A. culbersoni, A. diviornensis, A. hatchetti, A. healyi, A. polyphaga, A. rhysodes, etc. - uninucleated - irregular shape - acanthopodia(thorn-liked) 41 www2.le.ac.uk www.med-chem.com ww.labor-spiez.ch Trophozoite, 10-45 µm Cyst, 5-25 µm 43 11 16/07/64 Disease 1. Granulomatous Amoebic Encephalitis (GAE) 1. Granulomatous Amoebic Encephalitis (GAE) Symptoms - common in immunocompromised hosts - primary infection at the skin ulcer or lung, then . subacute (IP 8-30 days) with no specific symptoms spread hematogenously to brain . resemble viral, bacterial or tuberculosis meningitis - no evidence of infection via olfactory nerves 2. Acanthamoeba keratitis (AK) . mental status changes (86%), seizures (66%) - mostly in healthy persons (contact lens wearers) hemiparesis (53%), fever (53%), stiff neck 3. Cutaneous infection headache (53%), meningismus (40%), etc. - subacute granulomatous dermatitis - immunocompromised hosts 45 46 GAE GAE DOI: 10.5005/jp-journals-10028-1080 P. Singh, R. Kochhar, R.K. Vashishta, N. Khandelwal, S. Prabhakar, S. Mohindra and P. Singhi American Journal of Neuroradiology June 2006, 27 (6) 1217-1221; http://www.med-chem.com/para-site.php?url=org/acantham •The terms “ granulomatous” indicates hemorrhagic necrotizing GAE: spread hematogenously, possible distribute in frontal lobe,