Pathogenic Amoebae Be Able to Identify and Describe the Pathogenic Amoebae in Terms of Morphology, Life Cycle, Pathology, Symptoms, Transmission and Diagnosis

Home , Entamoeba histolytica, Sappinia pedata

16/07/64

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

Human Pathogen II

Assoc. Prof. Dr. Anchalee Wannasan

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

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)

Rapid movement by finger-like pseudopodia

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

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

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

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)

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, temporal lobe, parietal lobe, lesions or brain abscess (detected by neuroimaging scans) with likely through middle-cerebral artery (as these cortices are among the main regions supplied by

severe meningeal irritation and encephalitis. middle cerebral artery) Ong et al JCM, 2017 48 (NA Khan - Am J Infect Dis: 79-83, 2005) 12 16/07/64

Diagnosis GAE GAE- Acanthamoeba - 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 ruby.fgcu.edu 49

Treatment GAE 2. Acanthamoeba Keratitis (AK) - Brain surgery of multi focal areas is difficult.  a painful vision-threatening infection - combination drugs  Corneal ulceration, visual loss, and azoles drugs, pentamidine, blindness amphothericin B, chlorhexidine,  healthy person, esp. contact lens wearers rifampin, miltefosine, etc.  Developed countries (83% AK cases) - Most GAE cases were proven after death. (Carvalho et al. 2009)  Non-developed countries mostly related with agricultural-based activities

(Clarke and Niederkorn 2006; Gopinathan et al. 2009)

13 16/07/64

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 pathogens (Pseudomonas, Staphylococcus, Herpes, or Adenovirus, etc.)

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

(A) ring infiltration (most cases) • history of using contact lens (B) multiple ring infiltrates and hypopyon • confocal microscopy (C) near-total suppuration • corneal scraping, corneal biopsy from the infiltrate areas - staining / culture - IFA / specific PCR • unrecommended tests : Ab detection or amoebae isolation from lens case

(Barratt JLN., 2010) 14 16/07/64

Staining from corneal scrapings (Barratt JLN., et al, 2010) Vivo Confocal Microscopy

Calcofluor white Giemsa stain

Gram stain Giemsa stain

Treatment AK 3. Cutaneous infection . early infection: combination drugs + epithelial debridement • Vary clinical signs depended on underlying . combination drugs immunologic status. biguanides : chlorhexidine, polyhexamethylene biguanide (PHMB) • Both cysts and trophozoites diamidines : propamidine, hexamidine can be found on skin.

. late infection: permanent damage, drug resistance • very rare and self-limiting in immunocompetent host hypopyon, secondary infection  cornea transplantation

- 59 60

15 16/07/64

Cutaneous acanthamoebiasis

Lab Demonstration

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 62

Trichrome stain Uninucleated cyst

Iron-hematoxylin stain Glycogen vacuole

63 64

16 16/07/64

Trichrome stain Binucleated cyst

Fresh smear

65 66

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

10 µm

67 68

17 16/07/64

Naegleria fowleri Acanthamoeba castellanii Cyst Fresh smear from the culture 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 • uninucleated flagellate within 1 hr. at 37°C. • Thick-wall wrinkle ectocyst • The process is reversible. • Endocyst with various shapes e.g., polygonal , stellate, oval or spherical • Endocyst meet ectocyst at the ostioles (cyst pore).

69 70

Acanthamoeba castellanii Trophozoite Fresh smear from culture

5 µm

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

E-mail: [email protected] 71