Protozoa: Introduction and classification

Amoebae I. Pathogenic amoebae: Entamoeba histolytica,

Naegleria fowleri, Acanthamoeba spp.

Nimit Morakote, Ph.D.

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Protozoa-Simple classification

• Amoeba- pseudopodium(a)

• Flagellate- flagellum(a)

• Ciliate- cilia

• Sporozoa- spore, sex

• Microsporidium- microspore with polar tube • New classification= fungus

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Amoeba

• Trophozoite

–feeding stage, movement and feeding by pseudopodia

–Grow and multiply by binary fission

–1 nucleus

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• Cyst

– Trophozoite -> adverse environment -> stop feeding -> cyst wall -> cyst

– transfer stage, non-feeding

– resistant to environment

– Immature -> Mature, Infective cyst

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Pathogenic amoeba of man

–Intestinal amoeba

• Entamoeba histolytica

–Obligatory parasite

–Free-living amoeba (live in soil and water)

• Naegleria fowleri

• Acanthamoeba spp.

• Balamuthia mandrillaris

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• Human and primate = Definitive host

• Parasitize the large intestine

• Simple life cycle

Entamoeba histolytica

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Disease

• Amebic colitis -> amebic dysentery (Intestinal amoebiasis)

• Hematogenous spread

– to liver -> Amebic liver abscess

–other organs (e.g., lung, brain abscess)

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Stanley SL (2003) Lancet 7

Signs and symptoms

• Intestinal amoebiasis (บิดมีตัว) –Tenesmus, abdominal pain

–dysenteric stool (mucus with blood) fowl smell

• Amoebic liver abscess

–Fever, right upper quadrant abdominal pain

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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

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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.

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• 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.

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• 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

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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

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Permanent stain

• Nuclear characteristics: Concentric karyosome and even peripheral chromatin distribution

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Prevention & Treatment

• Prevention

–Boiled or filtered water

–Cook food

–Personal hygiene

• Treatment

–Metronidazole (Flagyl)

– Tinidazole

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Pathogenic free-living amoebae

Name Disease Affected organ

Onset Entry

Naegleria fowleri PAM Brain Acute Nose

Acanthamoeba GAE Brain Subacute

Skin/ lung

keratitis Eye Eye

Balamuthia mandrillaris

GAE Brain Subacute/ chronic

Skin/ lung

PAM, primary amoebic meningoencephalitis GAE, granulomatous amoebic meningoencephalitis

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Naegleria fowleri (Amoeboflagellate)

Trophozoite with lobopodia 10-15 m

Flagellate form

Cyst, 8-12 m 1 nucleus

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Naegleria fowleri

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Clinical manifestation

• Acute onset

• Stiff neck, sore throat, severe headache, vomiting, seizure, coma

• IP 3-7 days after exposure, dead within 10 days

Epidemiology

• Worldwide

• USA, 1962-2015= 138 known cases; mostly in southern part

• Swimming in lake or ponds with warm water

• Considered low risk

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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

Enflagellation test

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Prevention

• Chlorine in swimming pool 1-2 ppm

• Avoid swimming in natural pond especially winter to summer

• Wear nose clip

• Blow the nose

Treatment

• Miltefosine or combination anti-microbial therapy: amphotericin B, Azithromycine, rifampin, and azole drugs

• Intensive supportive care is required

• Only a few from hundred cases survive

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http://edition.cnn.com/2016/08/17/health/florida-brain-eating-amoeba/

http://edition.cnn.com/2016/08/23/health/brain-eating-amoeba-florida-teen-survives/

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Acanthamoeba

• Uninucleated trophozoites with acanthopodia, 25-40 m

www2.le.ac.uk

Cysts - polygonal, stellate, oval or

spherical endocysts, 15-20

m - 1 nucleus

• 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.

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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

Symptoms

–GAE

• Mental status changes, Seizures, Hemiparesis, Fever, Headache, Meningismus

• Subacute onset; IP 8-30 days

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http://www.emedicine.com/MED/topic10.htm

Acanthamoebic keratitis

–Keratitis

• Begins with a foreign-body sensation followed by pain, tearing, photophobia, blepharospasm, and blurred vision (IP only a few days)

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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

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Prevention

• Personal hygiene

• Avoid contact with soil

• Clean contact lens

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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

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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

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13-30 µm

Trophozoites- uninucleated

Cysts-uninucleated

15 to 60 µm in length

(http://www.eol.org/pages/2911550)

Enter human body via skin and respiratory route, then hematogenously spread to brain

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)

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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

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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%

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Case reports in Thailand

• PAM- 12 cases, First case report- B.E. 2525

– ศรีษะเกษ อุบลฯ นครปฐม สมุทรปราการ กรุงเทพฯ

• GAE- 11 cases

– อุบลฯ สุพรรณ กาญจนบุรี นครปฐม กรุงเทพฯ

• Acanthamoebic keratitis 5 cases

• BAE- 1 case motorcycle accident –pond -chronic nasal lesion

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

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End of lecture

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