Protozoa: Introduction and classification Amoebae I. Pathogenic amoebae: Entamoeba histolytica, Naegleria fowleri, Acanthamoeba spp. Nimit Morakote, Ph.D. 1
Protozoa-Simple classification Amoebapseudopodium(a) Flagellate- flagellum(a) Ciliate- cilia Sporozoa- spore, sex Microsporidiummicrospore with polar tube New classification= fungus 2
Amoeba Trophozoite feeding stage, movement and feeding by pseudopodia Grow and multiply by binary fission 1 nucleus 3
Cyst Trophozoite -> adverse environment -> stop feeding -> cyst wall -> cyst transfer stage, non-feeding resistant to environment Immature -> Mature, Infective cyst 4
Pathogenic amoeba of man Intestinal amoeba Entamoeba histolytica Obligatory parasite Free-living amoeba (live in soil and water) Naegleria fowleri Acanthamoeba spp. Balamuthia mandrillaris 5
Entamoeba histolytica Human and primate = Definitive host Parasitize the large intestine Simple life cycle 6 6
Disease Amebic colitis -> amebic dysentery (Intestinal amoebiasis) Hematogenous spread to liver -> Amebic liver abscess other organs (e.g., lung, brain abscess) Stanley SL (2003) Lancet 7 7
Signs and symptoms Intestinal amoebiasis (บ ดม ต ว) Tenesmus, abdominal pain dysenteric stool (mucus with blood) fowl smell Amoebic liver abscess Fever, right upper quadrant abdominal pain 8
Epidemiology Waterborne outbreak >325 water-associated outbreaks of parasitic protozoan disease North American and European outbreaks accounted for 93% G. duodenalis 40.6%, C. parvum 50.8%, E. histolytica 2.8%, C. cayetanensis 1.8%, T. gondii 0.9%, I. belli 0.9%, B. hominis 0.6% B. coli, microsporidia, Acanthamoeba and N. fowleri - 0.3%each Karanis P, Kourenti C, Smith H. Waterborne transmission of protozoan parasites: a worldwide review of outbreaks and lessons learnt. J Water Health. 2007 Mar;5(1):1-38. Review. 9 9
A survey reveals existence of both E. histolytica and E. dispar in the same area In general population, prevalence of E. dispar 10X more than E. histolytica Only about 10% of cyst carriers develop intestinal amoebiasis High prevalence among communities with poor socioeconomic conditions and sanitary level. 10
A survey in Phang-Nga province (Intarapuk A, et al, 2009) 455 fecal samples- 30 samples positive for cysts 12 (40%) E. histolytica 6 (20%) E. dispar 10 (33%) Mixed infection A survey in rural communities in Malaysia (Ngui R, et al, 2012) 63.5% E. histolytica; 19.2% E. dispar, 11.5% mixed infection 11
Diagnosis Intestinal amoebiasis Stool exam for trophozoites Fresh dysenteric stool -> smear in saline -> active movement with lobopodia (directional) Ingested red blood cells are suggestive Permanent stain Differentiate from E. dispar by immunological or molecular technique 12 12
Permanent stain Nuclear characteristics: Concentric karyosome and even peripheral chromatin distribution 13
Prevention & Treatment Prevention Boiled or filtered water Cook food Personal hygiene Treatment Metronidazole (Flagyl) Tinidazole 14 14
Pathogenic free-living amoebae Name Disease Affected organ Onset Entry Naegleria fowleri PAM Brain Acute Nose Acanthamoeba GAE Brain Subacut e Balamuthia mandrillaris keratitis Eye Eye GAE Brain Subacut e/ chronic Skin/ lung Skin/ lung PAM, primary amoebic meningoencephalitis GAE, granulomatous amoebic meningoencephalitis 15
Naegleria fowleri (Amoeboflagellate) Trophozoite with lobopodia 10-15 m Flagellate form Cyst, 8-12 m 1 nucleus 16
Naegleria fowleri 17
Clinical manifestation Acute onset Stiff neck, sore throat, severe headache, vomiting, seizure, coma IP 3-7 days after exposure, dead within 10 days 18
Epidemiology Worldwide USA, 1962-2015= 138 known cases; mostly in southern part Swimming in lake or ponds with warm water Considered low risk 19
Diagnosis History of swimming in natural ponds helps diagnosis CSF exam Hanging drop prep -> flagellate culture in nutrient agar -> colony -> trophozoites and cysts Autopsy Brain tissues- only trophozoite with large karyosome, no cyst 20
Enflagellation test 21
Prevention Chlorine in swimming pool 1-2 ppm Avoid swimming in natural pond especially winter to summer Wear nose clip Blow the nose 22
Treatment Miltefosine or combination antimicrobial therapy: amphotericin B, Azithromycine, rifampin, and azole drugs Intensive supportive care is required Only a few from hundred cases survive 23
http://edition.cnn.com/2016/08/17/health/florida-brain-eating-amoeba/ http://edition.cnn.com/2016/08/23/health/brain-eatingamoeba-florida-teen-survives/ 24
Acanthamoeba Uninucleated trophozoites with acanthopodia, 25-40 m www2.le.ac.uk Cysts - polygonal, stellate, oval or spherical endocysts, 15-20 m - 1 nucleus 25
Pathogenic to man A. castellani, A. culbersoni, A. diviornensis, A. hatchetti, A. healyi, A. polyphaga, A. rhysodes Found in environment: Air, fresh and sea water, soil, dust, polluted water around industrial area, Jacucci tubs, dental irrigation unit, etc. 26
Disease Granulomatous acanthamoebic encephalitis Occur mostly in debilitated or immunocompromised persons (Naegleriahealthy person) Primary infection at the skin (ulcer) or lung, then spread hematogenously, or via olfactory nerve Acanthamoebic keratitis (in healthy persons) Mostly in soft contact lens wearer 27
Symptoms GAE Mental status changes, Seizures, Hemiparesis, Fever, Headache, Meningismus Subacute onset; IP 8-30 days http://www.emedicine.com/med/topic10.htm 28
Acanthamoebic keratitis Keratitis Begins with a foreign-body sensation followed by pain, tearing, photophobia, blepharospasm, and blurred vision (IP only a few days) 29
Diagnosis Diagnosis CSF culture in nutrient agar seeded with intestinal bacteria Autopsy Brain tissues- both trophozoite (large karyosome) and cysts Keratitis Corneal scraping Stain or culture in nutrient agar seeded with E.coli 30
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Prevention Personal hygiene Avoid contact with soil Clean contact lens 32
Treatment GAE Combination antimicrobial therapy- Amphothericin B, Rifampin, etc. Miltefosine Keratitis 0.1% and miconazole nitrate 1% with neomycin or diamide (propamidine isethionate or hexamidine) with a cationic antiseptic (polyhexamethylene biguanide [PHMB] or chlorhexidine) for 1-12 months 33
Balamuthia mandrillaris Genetically related to Acanthamoeba Found in mandrill in San Diego Zoo, died of meningoencephalitis About 100 Cases of GAE- healthy people with emphasis on very young and very old age 34
Trophozoitesuninucleated 15 to 60 µm in length (http://www.eol.org/pages/2911 550) Enter human body via skin and respiratory route, then hematogenously spread to brain Cysts-uninucleated 13-30 µm 35
Epidemiology Infect both healthy and debilitated persons Contact with soil, organ transplant Risk factors People with HIV/AIDS, cancer, liver disease, or diabetes mellitus People taking immune system inhibiting drugs Alcoholics Young children or the elderly Pregnant women 100 cases worldwide (USA, Peru, Argentina, Brazil, Mexico, Argentina, Thailand) 36
Clinical manifestation Subacute or chronic disease (3 mo-2 yr) Initially headache, stiff neck, nausea, fever Followed by Headache, fever, nausea, mental state abnormalities, irritability, hemiparesis, cranial nerve palsies, hallucinations, photophobia, sleep and speech disturbance, and seizures May have unusual skin lesions that persist over months 37
LAB DIAGNOSIS Wet mount of CSF for amoebae In vitro culture in mammalian cell monolayer Autopsy- brain section, speciation by anti- Balamuthia serum Treatment: combination anti-microbial therapy Prognosis: Extremely poor, mortality rate >98% 38
Case reports in Thailand PAM- 12 cases, First case report- B.E. 2525 ศร ษะเกษ อ บลฯ นครปฐม สม ทรปราการ กร งเทพฯ GAE- 11 cases อ บลฯ ส พรรณ กาญจนบ ร นครปฐม กร งเทพฯ Acanthamoebic keratitis 5 cases BAE- 1 case motorcycle accident pond -chronic nasal lesion 39
Demonstration E. histolytica permanent stain: observe nuclear characteristics Preserved specimens: Iodine stain and search for E. histolytica by microscope Acanthamoeba wet mount: observe trophozoite and cyst morphology 40
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