Pathogenic Amoebae

Human II

Assoc. Prof. Dr. Anchalee Wannasan

1 Objectives • be able to identify and describe the pathogenic amoebae in terms of morphology, life cycle, pathology, symptoms, transmission and diagnosis histolytica

• Disease: • Distribution: worldwide - second leading cause of death after malaria - developing countries: poor areas - developed countries : immigrants or travelers from endemic areas • Transmission: - fecal-oral route - venereal transmission (homosexuals) • 2 stages in the life cycle

Cyst (dormant form) Trophozoite (active form)

• Infective satge: mature with 4 nuclei www.medical-labs.net

karyosome

Chromatoid body

Peripheral chromatin www.medical-labs.net 5 Cyst ~10-20 µm

• round or oval shape • thin and tough cyst wall • contains 1, 2 or 4 nuclei • centric karyosome • peripheral chromatin • chromatoid bar with rounded ends (Cigar-like) 7

Trophozoite ~15-60 µm

• irregular shape • a round nucleus • small, centric karyosome • peripheral chromatin scattered evenly throughout the inner nuclear membrane • fine granular endoplasm ( vacuoles + RBC) 10 Rapid movement by finger-like 12 Pathology and symptoms • Amoebiasis is a disease caused by E. histolytica. • Forms of amoebiasis 1) Intestinal amoebiasis - 90% is asymptomatic cyst passers (E. dispar ?) - 10% is symptomatic (Invasive amoebiasis) 2) Extraintestinal amoebiasis 1. Intestinal amoebiasis (Invasive amoebiasis)

❑ incubation period is variable, (mostly 1-4 w) ❑ characterized by 4 markers:

o amoebic (mucus and bloody )

o trophozoites with ingested RBC (, biopsy)

o amoebic (endoscopy) o positive serological test for specific Ab ❑ Other clinical manifestations

o increasing diarrhea progressing to mucous-bloody o lower , tenesmus o , 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 - Amoeboma (immune response to trophozoites)

15 Ameboma

https://www.wjgnet.com/1007-9327/full/v22/i36/8234.htm

• Granulation tissue and fibrotic thickening caused by

host immune response to trophozoites, Fig A, B) 16 Amebic colitis

http://intranet.tdmu.edu.ua/data/kafedra/internal/med_biologia/classes_stud/en/med/prophylactic%20medicine/ptn/medical%20biology/1%20course/theme%2008.htm • Severe dysentery with multiple ulcers in the large bowel, and a bloody diarrhea

17 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) o rounded and small ulcer with slightly elevated rims o necrotic center - appears dimpled or hemorrhagic - filled with yellowish mucous 18 2) Irregular lesion (1- 5 cm in length)

o shallow but broad with elevated rims o filled with fibrin and yellow exudates

https://www.researchsquare.com/article/rs-29990/v1

Irregular cecum 19 lesions Flask-shape amoebic ulcer

4.bp.blogspot.com

20 Pathogenesis

• glandular hyperplasia • stromal edema • atrophy of villi & epithelium • infiltration of neutrophils • deep into submucosa • 2nd bacterial

21 (flask-shape amoebic ulcer) 2. Extraintestinal amoebiasis

• Hematogenously spread to and other organs (lung, pericardium, brain, skin, and genitourinary tract, etc.) • Liver is the most common involvement site • 3–9 % of all intestinal amebiasis cases develop to amoebic (ALA) Amoebic liver abcess (ALA) ▪capillary obstruction in portal system () ▪ 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 Rupture of abscesses into the abdomen or chest may lead to death. Amoebic liver https://www.sciencephoto.com/media/250192/view/liver-abscess

https://en.wikipedia.org/wiki/Amoebic_liver_abscess

abcess

d_Pathogenesis/figures?lo=1 https://www.researchgate.net/publication/221929027_Amoebiasis_in_the_Tropics_Epidemiology_an Diagnosis

 Intestinal amebiasis

• Microscopic examination (stool or rectal smear) - dysenteric stool : trophozoites - nondysenteric stool : - from E. dispar using isozyme analysis, serological methods, PCR • Biopsy from colonoscopy / sigmoidoscopy

25 • Extraintestinal amebiasis

• stool exam may not be helpful • aspirates from abscess may not find amoebae • biopsy from liver abscess rims (recommended) • imaging procedure (ultrasound/ CT / MRI) • serological methods (ELISA) - discriminate ALA from pyogenic liver abscess - negative in Entamoeba dispar infected patient • PCR

26 http://medicinembbs.blogspot.com/2013/01/amoebic-liver-abscess.html

Charateristics of pus from ALA: anchovy paste-liked color without smell Treatment

 drugs of choice : - effective to both intestinal and tissue amoebiasis derivatives such as , and  ALA - Percutaneous drainage or surgical aspiration  E. dispar infection does not require the treatment

28 Pathogenic free-living amebae

o Primary amoebic meningoencephalitis (PAM)  spp. o Granulomatous amoebic encephalitis (GAE) o Acanthamoebic keratitis (AK)  o GAE  pedata o GAE

29 Common features (pathogenic FLA)

• facultative free-living parasites • generally found in the environment • mortality rate approach 100% • no specific therapeutic agents

30 Naegleria fowleri

• known as the brain-eating • Distribution : worldwide (water and soil resources) • Disease : primary amebic meningoencephalitis most cases were immunocompetent host • Most clinical isolates are thermophilic (> 45°C). • 3 stages in LC: cyst , trophozoite and flagellate (Amoeboflagellate)

31 Morphology Cyst ~ 8-20 µm

https://www.si.mahidol.ac.th/th/department//articledetail.asp?ac_id=7&pageno=1 • round with double cyst walls • big karyosome surrounded by halo • never found cyst stage in host tissue Trophozoite Flagellate ~ 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 Life cycle

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 Clinical manifestation

 Incubation period: 3-7 days

 acute common cold-like symptoms: fever, rhinitis, stiff neck, sore throat and severe headache similar to that of acute bacterial and viral meningitis

 Rapidly progression with high fever, vomiting, seizure, edema of lung and brain, respiratory failure, coma  Death within 10 days

37 Diagnosis

 immunocompetent host (mainly children & young adult)  history of water exposure (exp. swimming)  CSF sedimentation : found only trophozoite o culture on non-nutrient agar with gram (-ve) o enflagellation test, PCR • brain tissues: found only trophozoite o staining o IFA, Immunohistochemistry o PCR Treatment

 no specific therapeutic agents

 Combination of available drugs which can cross - brain barrier

or combination anti-microbial therapy: amphotericin B, Azithromycin, rifampin, and azole drugs

 Intensive supportive care is required

 Only a few from hundred cases survive

39 Prevention

 Chlorine in swimming pool 1-2 ppm

 Avoid swimming in fresh natural resources

 Wear the nose clip during swimming

 Blow the nose using sterile reagent

40 Acanthamoeba

• Free-living protozoan (facultative) parasites - worldwide distribution : soil, air, dust, fresh and sea water, Jacucci tubs, dental irrigation unit, contact lens, etc. - usually concentrate at the bottom of the lake

• Pathogenic to man (thermophilic) • Exp. A. castellani, A. culbersoni, A. diviornensis, A. hatchetti, A. healyi, A. polyphaga, A. rhysodes, etc.

41 Morphology

A) Cyst ~ 5-25 µm

- uninucleated - thick-wall polygonal , stellate, oval or spherical endocyst with ostioles - wrinkle ectocyst ~ 10-45 µm B) Trophozoite

- uninucleated - irregular shape - acanthopodia (thorn-liked) www2.le.ac.uk www.med-chem.com

ww.labor-spiez.ch

Trophozoite, 10-45 µm Cyst, 5-25 µm 43

Disease 1. Granulomatous Amoebic Encephalitis (GAE) - common in immunocompromised hosts - primary infection at the skin ulcer or lung, then spread hematogenously to brain - no evidence of infection via olfactory nerves 2. (AK) - mostly in healthy persons (contact lens wearers) 3. Cutaneous infection - subacute granulomatous dermatitis - immunocompromised hosts

45 1. Granulomatous Amoebic Encephalitis (GAE)

Symptoms ▪ subacute (IP 8-30 days) with no specific symptoms ▪ resemble viral, bacterial or tuberculosis meningitis

▪ mental status changes (86%), seizures (66%) hemiparesis (53%), fever (53%), stiff neck headache (53%), meningismus (40%), etc.

46 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; •The terms “ granulomatous” indicates hemorrhagic necrotizing lesions or brain abscess (detected by neuroimaging scans) with severe meningeal irritation and encephalitis. (NA Khan - Am J Infect Dis: 79-83, 2005) GAE

http://www.med-chem.com/para-site.php?url=org/acantham

GAE: spread hematogenously, possible distribute in frontal lobe, temporal lobe, parietal lobe, likely through middle-cerebral artery (as these cortices are among the main regions supplied by middle cerebral artery) Ong et al JCM, 2017 48 Diagnosis GAE

- difficult (similar symptoms as other CNS infection) - historical exposure to water or soil - CT, MRI : not specific - brain biopsy o staining (trophozoites and cysts) o IFA, culture, PCR - CSF o rarely find amoebae by microscopy o culture, PCR - Serological test : may not be useful

49 GAE- Acanthamoeba

ruby.fgcu.edu Treatment GAE - Brain surgery of multi focal areas is difficult. - combination drugs azoles drugs, pentamidine, amphothericin B, chlorhexidine, rifampin, miltefosine, etc. - Most GAE cases were proven after death.

51 2. Acanthamoeba Keratitis (AK)

 a painful vision-threatening infection  Corneal ulceration, visual loss, and blindness  healthy person, esp. contact lens wearers  Developed countries (83% AK cases)

(Carvalho et al. 2009)  Non-developed countries mostly related with agricultural-based activities

(Clarke and Niederkorn 2006; Gopinathan et al. 2009) Daniel W. Clarke and Jerry Y. Niederkorn Symptoms AK ➢ Incubation period : a few days to several weeks - begins with a foreign-body sensation - tearing, epithelial defects and photophobia - inflammation with redness, stromal infiltration, stromal opacity with extreme pain (radial neuritis), stromal abscess and finally blindness

➢ Frequent misdiagnosis to other keratitis from common (Pseudomonas, Staphylococcus, Herpes, or Adenovirus, etc.)

54 Clinical AK appearance (Barratt JLN, et al., 2010)

(A) ring infiltration (most cases) (B) multiple ring infiltrates and hypopyon (C) near-total suppuration

(Barratt JLN., 2010) Diagnosis

• history of using contact lens • confocal microscopy • corneal scraping, corneal biopsy from the infiltrate areas - staining / culture - IFA / specific PCR • unrecommended tests : Ab detection or amoebae isolation from lens case Vivo Confocal Microscopy Staining from corneal scrapings (Barratt JLN., et al, 2010)

Calcofluor white Giemsa stain

Gram stain Giemsa stain Treatment AK ▪ early infection: combination drugs + epithelial debridement ▪ combination drugs biguanides : chlorhexidine, polyhexamethylene biguanide (PHMB) diamidines : , hexamidine

▪ late infection: permanent damage, drug resistance hypopyon, secondary infection → cornea transplantation

- 59 3. Cutaneous infection

• Vary clinical signs depended on underlying immunologic status.

• Both cysts and trophozoites can be found on skin.

• very rare and self-limiting in immunocompetent host

60 Cutaneous acanthamoebiasis

https://www.labce.com/spg931637_illness_and_symptoms_continued_acanthamoeba_specie.aspx • Hematogenous spreading to other tissues in immunocompromised host • Involvement of CNS leads to death within weeks • early treatment : topical combination drugs (antimicrobials and azoles drugs) or surgery

61 Lab Demonstration

62 Glycogen vacuole

63 Trichrome stain Uninucleated cyst

Iron-hematoxylin stain

64 Trichrome stain Binucleated cyst

Fresh smear

65 66 10 µm

67 Naegleria fowleri Trichrome stain Cyst

• ~ 8-20 µm in diameter • round with unstained double cyst walls • big karyosome surrounded by halo • never found cyst stage in host tissue

68 Naegleria fowleri Fresh smear from CSF Trophozoite Lobopodia • ~ 10-35 µm , irregular shaped • big karyosome surrounded by halo • often seen in host tissue

Flagellate Trichrome stain • ~ 10-16 µm, pear-shaped flagellum • In depriving condition such as distilled water, trophozoite can transform into flagellate within 1 hr. at 37°C. • The process is reversible.

69 Acanthamoeba castellanii Cyst Fresh smear from the culture

• uninucleated • Thick-wall wrinkle ectocyst • Endocyst with various shapes e.g., polygonal , stellate, oval or spherical • Endocyst meet ectocyst at the ostioles (cyst pore).

70 Acanthamoeba castellanii Trophozoite Fresh smear from culture

5 µm

• uninucleated • irregular shape • acanthopodia (thorn-liked)

E-mail: [email protected] 71