Protozoa: Introduction and classification Amoebae I. Pathogenic amoebae: Entamoeba histolytica, Naegleria fowleri, Acanthamoeba spp.

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1 Protozoa: Introduction and classification Amoebae I. Pathogenic amoebae: Entamoeba histolytica, Naegleria fowleri, Acanthamoeba spp. Nimit Morakote, Ph.D.

2 Protozoa-Simple classification Amoeba- pseudopodium(a) Flagellate- flagellum(a) Ciliate- cilia Sporozoa- spore, sex Microsporidium (fungus)- microspore with polar tube

3 Amoeba Trophozoite feeding stage, movement and feeding by pseudopodia Grow and multiply by binary fission 1 nucleus

4 Cyst Trophozoite -> adverse environment -> stop feeding -> cyst wall -> cyst transfer stage, non-feeding resistant to environment Immature -> Mature, Infective cyst

5 Pathogenic amoeba of man Intestinal amoeba Entamoeba histolytica Obligatory parasite Free-living amoeba (live in soil and water) Naegleria fowleri Acanthamoeba spp. Balamuthia mandrillaris

6 Entamoeba histolytica Human and primate = Definitive host Parasitize the large intestine Simple life cycle 6

7 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

8 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

9 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 Mar;5(1):1-38. Review. 9

10 A survey reveals existence of both E. histolytica and nonpathogenic 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.

11 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

12 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

13 Permanent stain Nuclear characteristics: Concentric karyosome and even peripheral chromatin distribution

14 Prevention & Treatment Prevention Boiled or filtered water Cook food Personal hygiene Treatment Metronidazole (Flagyl) Tinidazole 14

15 Pathogenic free-living amoebae Name Disease Affected organ Onset Entry Naegleria fowleri PAM Brain Acute Nose Acanthamoeba GAE Brain Subacute Skin/ lung Balamuthia mandrillaris keratitis Eye Eye GAE Brain Subacute/ chronic Skin/ lung PAM, primary amoebic meningoencephalitis GAE, granulomatous amoebic meningoencephalitis

16 Naegleria fowleri (Amoeboflagellate) Trophozoite with lobopodia m Flagellate form Cyst, 8-12 m 1 nucleus

17 Naegleria fowleri 17

18 Clinical manifestation Acute onset, IP 3-7 days after exposure, dead within 10 days Stiff neck, sore throat, severe headache, vomiting, seizure, coma 18

19 Epidemiology Worldwide USA, = 138 known cases; mostly in southern part Swimming in lake or ponds with warm water Considered low risk

20 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 stage 20

21 Enflagellation test

22 Prevention Chlorine in swimming pool 1-2 ppm Avoid swimming in natural pond especially winter to summer Wear nose clip Blow the nose 22

23 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 in-eating-amoeba-florida-teen-survives/ 23

24 Acanthamoeba Uninucleated trophozoites with acanthopodia, m Cysts - polygonal, stellate, oval or spherical endocysts, m - 1 nucleus 24

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. 25

26 Disease Granulomatous acanthamoebic encephalitis Occur mostly in debilitated or immunocompromised persons (Naegleria- healthy 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 26

27 Symptoms GAE Mental status changes, Seizures, Hemiparesis, Fever, Headache, Meningismus Subacute onset; IP 8-30 days 27

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 and prvention 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 Prevention Personal hygiene Discard and replace contact lens case monthly or, at a minimum, every three months 29

30 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 30

31 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 31

32 Trophozoites- uninucleated 30 to 60 µm in length with filamentous characteristic ( Enter human body via skin and respiratory route, then hematogenously spread to brain Cysts-uninucleated With wavy ectocyst µm 32

33 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)

34 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 Likely to take a cutaneous route before secondarily attacking the CNS, in 5-8 months

35 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%

36 Case reports in Thailand PAM- 12 cases, First case report- B.E 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 36

37 End of lecture