Parasitology

Free Living Amoebas & Ciliates (Laboratory)

Parasitology Department’s PPT |

Kingdom Protista
Protozoa
Generalities
1. Environment is wet born for feeding, locomotion, osmoregulation, and
reproduction.
2. Vegetative state: Cyst Form
3. Feeding state/Infective state: Trophozoite form

General Classification of Class Protozoans Infecting Humans

Phylum
SARCODINA – Pseudopodia
(Hyaline foot-like extrusions)
MASTTIGOPHORA – Flagella
structures (whip-like structures)
Organism
• Entamoeba histolytica
• Acanthamoeba
• Naegleria Fowleri in trophozoite
form
• Balamuthia mandrillaris - not in Protozoa: Microspora
the book • Coiled polar filament
• Giardia Lamblia
• Trichomonas Vaginallis
• Leishmania donovani
• Naegleria Fowleri in Flagellate
form – not in the book

CILIOPHORA – Cilia (Hair-like • Balantidium Coli

projections)
APICOMPLEXA – apical complexes • Babesia
• Plasmodium
MICROSPORA - Spore-forming • Microsporidum
parasites (related to fungi but not
classified on kingdom fungi
Basic Compositions
1. Nucleus and Nuclei
2. Cytoplasm
3. Outer limiting membrane
4. Organelles
General Locomotion
1. Cilia
2. Pseudopodia
3. Flagella

Protozoans associated with Clinical Practice

1. Intestinal Amoeba
2. Commensal Amoeba
Protozoa: Apicomplexa 3. Free-Living Pathogenic Amoeba
Penetration 4. Ciliates and Flagellates
• Apical complexes 5. Coccidian
6. Other Intestinal Protozoans
7. Plasmodium
8. Babesia
9. Blood and Tissue Flagellates

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 Parasitology
Free Living Amoebas & Ciliates (Laboratory)
Parasitology Department’s PPT |

10. Microsporidia 2. Feeding Methods: Food Cup Sucking and Pinocytosis/Phagocytosis

3. Replication: Mitosis
Free Living pathogenic Amoebas/ Opportunistic pathogens 4. Movement: Sluggishly with Polydirectional - 50um/min
1. Associated Amoebic Protozoa that has the capacity to survive and 5. Infective Stage
reproduce of their own biological metabolism outside (environment) or
inside the (Host).
2. Considered as unicellular protozoa
Acanthamoeba
• A. castellanii
• A. polyphaga
• A. culbertsoni
Generalities
1. Active Form /Feeding Stage - Trophozoite Stage
2. Dormant Form - Cyst Stage
3. Reproduction - Binary Fission
4. Isolated - Air, Aquaria, Soil, Mineral Water, Deep/Sea Well Water,
Swimming/Medicinal Pools, Contact Lens Cleaning Solution / Amoebic
Keratitis
5. Targets: Eyes and Brain, Systemic (Immunocompromised) Occurs when the environment is optimal for growth
a. Slender Spine-Like Acanthapodia
b. Usually Pleomorphic shape, Round to Oval Shape (depends on the
movement.
c. Measures: 15-45um
d. Nucleus is located center cell
e. Protected by Plasma Membrane
f. Inside Filled with Cytoplasm
g. Lobopodium - Fan-shaped pseudopodium
h. Spinous Process – Cone shaped and spiculate process

PYGC (Proteose-Peptone, yeast extract, glucose + cysteine) medium

Life Cycle Reproduction – fissiparity cycles (10 hours or 6-24 hours)

Binary Fission – Occurring mitosis phases

Trophozoite to Cyst stage

1. Resistant to harsh environment - 10-24 Hours to Form
2. If loss of nutrients in environment
3. Infective Stage
4. Low Metabolic Rate
5. Mobility: Non-Motile

Routes: Occurs in Unfavorable Environment: Desiccation, Low 02, and Low Nutrient
a. Eyes Membranes - Keratitis a. Spherical Shape = Looses the Pseudopodium and Spinous Process
b. Nasal Membranes Lower Respiratory Tract - GAE or Disseminated Disease b. Round and Oval Shape
c. Broken Skin = Skin Lesion c. Measures: 10-25um
Stages: d. Ostioles (Microcrater Like) = Area where the excysting trophozoite will exit
a. Cyst and serve to monitor the external environment
b. Trophozoite e. Dense Double Walled Structure - Separated with TRANSPARENT BAND
SPACE
Infective: Cyst and Trophozoite Stages f. Inner Wall/ Endocyst - Smooth Polyhedron – Triangle/Polygonal/Circle/Star
Diagnostic: Cyst and Trophozoite Stages g. Outer Wall/Ectocyst = Fibrous Wrinkled Material
Morphology
Trophozoite stage
1. These feeds on Gram Negative Bacteria, Blue Green Algae, and
Yeast/Fungi

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 Parasitology
Free Living Amoebas & Ciliates (Laboratory)
Parasitology Department’s PPT |

Cyst stage

Cyst to Trophozoite Excystation

Granulomatous Amoebic Encephalitis (GAE)

Clinical Manifestations
Amoebic Keratitis
• Corneal epithelium opacity
• Associated with use of contaminated contact lenses and patients with
immunosuppression or history of eye trauma
• Contact Lenses - Poor Hygiene and Poor disinfection practices
• Location - Corneal Epithelium
• Co-Infection - With other Organisms
• Corneal Ring Infiltration, Corneal Ulcer, Keratoneuritis
• Incubation period – 10 days
• Presence of the Amoeba in the BRAIN (Parenchyma and Blood Vessel)
tissue
Destructive Encephalopathy + Meningeal Irritation
• Associated in patients with Immunosuppressive cases
• Headache, Fever, Seizures, Coma, Hallucinations, Paralysis, Increase
ICP
• SKIN LESIONS = Diagnostic clue

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 Parasitology
Free Living Amoebas & Ciliates (Laboratory)
Parasitology Department’s PPT |

CSF with Giemsa

Brain edema, deformation and herniation, necrosis in Balamuthia mandrillasis

(both Acanthamoeba spp. And Balamuthia mandrillaris can cause GAE)

Diagnostic Tests PCR – Detection of Acanthamoeba cysteine proteinase gene (AcCP) and
from EH
1. GAE - During Autopsy – Occurred on severe cases in Immunosuppressed
Treatment
patients
1. 5-Flurocytosine
2. Using H&E per Tissue Biopsy (Brain) - Autopsy
2. Ketoconazole
3. CSF - Culture (Early Diagnosis)
3. Itraconazole
4. Acanthamoeba Keratitis - Corneal Tissue Biopsy
4. Pentamidine
5. PCR using specific primer pairs - Detection of CYSTEIN PROTEINASE Gene
5. Amphotericin B
6. Clotrimazole + Pentamidine + Isothionate + Neosporin - Acanthamoeba
Keratitis
7. Avoid using topical corticosteroids - Corneal Keratitis

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 Parasitology
Free Living Amoebas & Ciliates (Laboratory)
Parasitology Department’s PPT |

Balamuthia Mandrillaris Stages:

Generalities a. Cyst
1. Leptomyxid Amoeba - smaller branching projections b. Trophozoite
2. Replication: Mitosis in trophozoite form
3. TARGET Infective Stages: Trophozoite and Cyst Stages
• BRAIN = GAE = FATAL Diagnostic Stages: Cyst and Trophozoite stages
• Disseminated Disease Morphology
• Skin Lesions 1. This amoeba CANNOT be distinguished with ACANTHAMOEBA Sp. - On
4. Location: Dust and Soil, Water routine Wet-Mount histology
2. Electron Microscopy may showcase differentiation
3. Antigen-Antibody test for Monoclonal and Polyclonal Antisera can be used
for Direct Fluorescent Antibody Assay / Immunoperoxidase Assays

Cyst Stage
1. Can be seen histologically in tissue specimens
2. Spherical in shape
3. Measures: 6-30 um
4. Solitary Nucleus with (2) Nucleoli
5. Composed of 2 walls separated by a transparent space
a. Exocyst - Wrinkled outer wall
b. Endocyst - Smooth inner wall (can be polygonal, starshaped)
*Very similar with Acanthamoeba morphology

1st Reported study of Balamuthia Mandrillaris in 1986 by Mandril Baboon

Life Cycle

Trophozoite stage
1. Can be seen histologically in tissue specimens
2. Pleomorphic with Leptomyxoid Character (Culture) = LONG SLENDER
PSEUDOPODIA
3. Measures: 12-60 um
4. Solitary nucleus or Binuclear with centrally located Nucleoli (2-3)
*Very Similar with Acanthamoeba morphology

Routes:
a. Nasal Membranes Lower Respiratory Tract - GAE or Disseminated Disease
• Can be inhalation = AIRBORNE Cyst Forms
• Swimming can be also associated (Trophozoite) - Not Mentioned in
CDC
b. Broken Skin = GAE or Disseminated Disease
• Immunocompetent and Immunocompromised patients
• Direct inoculation

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 Parasitology
Free Living Amoebas & Ciliates (Laboratory)
Parasitology Department’s PPT |

Treatment
1. Pentamidine IV
2. Sulfadiazine Oral
3. Flucytosine Oral
4. Fluconazole Oral or IV
5. Azithromycin Oral or IV or Clarithromycin
6. Miltefosine Oral
Clinical Manifestations
7. Other Supporting Agents
Granulomatous Amoebic Encephalitis (GAE) & Disseminated disease
Naegleria fowleri
1. Seen in both Immunocompromised and Immunocompetent patients
• N. gruberi – Non-pathogenic
2. Very Difficult to diagnose using routine H&E histology for speciation against
• N. fowleri - Pathogenic
other disease-causing GAE.
• N. philippinensis – Local
3. Only IF staining, Serologic Test by CDC, PCR Methods can distinguish the
Generalities
pathogen.
1. Considered to be a FREE LIVING PATHOGENIC AMOEBO-FLAGELLATE
4. AUTOPSY SPECIMENS: Cyst and Trophozoite are noted in the Biopsy
2. Located on the FRESH WATER and SOIL
5. RECOMMENDED: https://www.cdc.gov/parasites/balamuthia/audience-
3. Single Celled Amoeba that feed for Bacteria
hcp.html
4. Mode of ENTRY Is NASAL TISSUE > OLFACTORY NERVES > BRAIN
6. Clinical: Rhinitis, Skin Infections, CNS Signs, Infection Signs
5. Cases the Fatal Form of MENINGOENCEPHALITIS (PAM = Primary Amoebic
Meningoencephalitis) = ALWAYS FATAL!!
6. Layman's Term: BRAIN EATING AMOEBA
7. Thermophilic

Diagnostic Tests
• CSF Findings
1. Lymphocytic Pleocytosis
2. Normal or Low Glucose concentration
3. Elevated Protein concentration
4. Amoeba is difficult to visualize in CSF via Wet Mount Microscopy (1 vs.
109 Cases) - RARE (Cyst or Trophozoite)
*Naegleria Sp. = Numerous Amoeba seen in CSF (PAM)
6. PCR or IF Confirmation is REQUIRED.

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 Parasitology
Free Living Amoebas & Ciliates (Laboratory)
Parasitology Department’s PPT |

b. Swimming Form = Flagellate Form

c. Resistant Form = Cyst Form

Infective stage: Trophozoite

1st case of infection 1965 by Dr. Malcom Fowler
Diagnostic stage: Trophozoite
Morphology
Trophozoite Form
1. Infective stage
2. Shaped like a Lobose Monopseudopodium = BROAD THICKENED
PSEUDOPODIA = LOBOPODIUM
3. If Rounded = 10-15um
4. Single Prominent Nucleus - with a centrally located nucleolus
5. Cytoplasm = Granular
6. Measures: 10-35um
7. Replication: Binary Fission at 35 C degree temperature
8. Motility: FAST = 1um/sec (LINEAR FORWARD DIRECTION)
9. In the Brain/Human Host = No Cyst form
10. HEAT RESISTANT = 45 C degree
11. Chorine water pools - will not kill the Amoeba

Life Cycle

Flagellate Form
1. Presence of (2) flagella at one end of the pole
2. The body is cigar/pear shaped body
3. Usually Transforms in this morphology if the environment changes - Distilled
Water
4. THIS IS THE DIFFERENTIAL DIAGNOSIS During Culture = Other Free-Living
Amoebas do not transform into Flagellated Type VS. Acanthamoeba

Cyst Form
1. Only occurs in harsh environment like cold temperatures and loss water
2. Usually NOT seen in Autopsy specimens in human host
3. Measures - 8-12 um
4. Monolayer Outer Coating
Routes:
Mode of ENTRY Is NASAL TISSUE > OLFACTORY NERVES > BRAIN
Stages:
a. Amoeba Form = Trophozoite Form

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 Parasitology
Free Living Amoebas & Ciliates (Laboratory)
Parasitology Department’s PPT |

Treatment
1. Miltefosine - Anti-Leishmania agent = Recommended by the CDC (Latest)
2. Older: Amphotericin B, Azithromycin, and Clotrimazole agents
3. Supportive Therapy - Dexamethasone etc.

Clinical Manifestations
Primary Amoebic Meningoencephalitis (PAM)
1. FATAL
2. 5-7 Days with/ without intervention is FATAL
3. There are Gastritis and Diarrhea then CNS Signs
4. Others: Fever, Headache, Vomiting, Coma, and Death
5. Route: Intranasal or Oral (?) some authors
6. Dust-Born Cyst in Aerated = can be also possible cause

Naegleria fowleri
• 3 only survive for 150 cases in 50 years

Brain Inflammation & Destruction Ciliates

Balantidium Coli
Generalities
1. Classified as CILIOPHORA - Presence of Cilia at Trophozoite Stage (The one
and only protozoan that can infect humans)
2. Disease: BALANTIDIASIS INFECTION - affects the intestinal epithelium causing
ulcers and bleeding
3. Associated with exposure handling history with Pigs
4. Other history can be associated as well in fomite cross-contaminations or
with poor hygiene
Life Cycle

Brain Frontal Cortex

Under IF
Diagnostic Tests
• CSF
1. CSF Sample - Should not be refrigerated = Kills the Amoeba
(Thermophilic)
2. Sediment - Wet mount
3. Culture Studies • Primary reservior host - pigs
• 2 important reserviors host = human and pigs
• Excystation - Occurs upon the ingestion towards in the small
intestine (Release of trophozoite)
• The Trophozoite colonize (transverse binary fission) the intestines
(Small, Large Intestines)
• Encystation - Trophozoite become cyst as it stays on the intestine
• Passed in the Feces - Cyst and Trophozoites
Routes:
Mouth (Ingestion)
Stages:
a. Cyst
b. Trophozoite

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 Parasitology
Free Living Amoebas & Ciliates (Laboratory)
Parasitology Department’s PPT |

Infective stage: Cyst form via Ingestion of Fomite, Water, Food Cyst Form
Diagnostic stage: Cyst and Trophozoite Forms 1. MEASURES: 40-60um
2. Spherical to Ovoid in Shaped
3. Cyst wall - Thickened
4. Encysted Ciliate - The cilia is found protected by the cyst wall coating
5. Infective stage for Transmission = Ingestion

Morphology
Trophozoite Form
1. Measures: 30-300um x 30-100 um
2. Cystosome - Apical portion entry of food
3. Cytopyge - Opposite pole to which excretion occurs
4. Macronucleus - Sausage/Bean shaped
5. Micronucleus - Pointed shaped
Clinical Manifestations
6. Contractile Vacuole – 2pcs= 2
Balantidiasis (Balantidiak Dysentry)
7. Mucocyst - Located beneath the cell membrane that acts for adhesion
1. Trophozoite attaches to the intestinal mucosa and adhere
8. Cilia - Present on the Plasma Membrane
2. Release Hyaluronidase
9. No Cyst Wall
3. Immune reaction may occur leading to mucosal damage of ulcer
10. Capable for Replication - Transverse Binary Fission
formation and bleeding
11. Capable of Tissue Destruction in intestinal mucosa
4. Complication: Perforation, Acute AP, Spread of Infection (Extra-Intestinal)
CROSS: Rounded Base with widened neck

20 y/o, Male

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 Parasitology
Free Living Amoebas & Ciliates (Laboratory)
Parasitology Department’s PPT |

Diagnostic Tests

Wet Mount
Treatment
1. Tetracycline
2. Metronidazole
3. Iodoquinol
4. Supportive

References:
Parasitology Dept. PPT
-Henry’s Clinical Diagnosis and Management by Laboratory Methods
-Medical Parasitology in the Philippines
-CDC
-Acanthamoeba Keratitis: Diagnosis and Treatment

Use at your own RISK!!!

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