AMOEBA

Classification of Amoeba

-​ Is a single-celled protozoa that constantly changes its shape

-​ “amoeba” from the Greek word “amoeboid” meaning “change”
-​ They constantly change their shape due to the presence of an organ of locomotion called “pseudopodium”

Classification Based on Habitat

-​ Are classified as intestinal amoebae and free-living amoebae

-​ Intestinal amoebae: inhabitat in the large intestine of humans and animals
-​ Free-living amoebae: are small free living and opportunistic pathogens

Taxonomical Classification

-​ Traditionally, amoeba was classified in the 1980s within the Phylum Sarcomastigophora.
-​ Specific classifications included
-​ ​ Subphylum: Sarcodina,
-​ ​ Superclass: Rhizopoda,
-​ ​ Class: Lobosea,
-​ ​ Subclass: Gymnamoebia,
-​ ​ Order: Amoebida,
-​ ​ Family: Endamoebidae.
-​ In the last 30 years, molecular techniques have updated the taxonomy.

PATHOGENIC INTESTINAL AMOEBA

Entamoeba histolytica
Introduction -​ More common in tropical and subtropical countries
-​ Has three subspecies – histolytica, dispar, and moshkovskii
-​ Cysts and trophozoites of all three subspecies are morphologically
indistinguishable
-​ Is the pathogenic species causing amoebic dysentery and a wide range of other
invasive diseases (amoebic liver abscess)
History -​ Was first described by Fedor Losch (1875) from Russia
-​ The species name was first coined by Fritz Schaudinn in 1903
-​ Brumpt described the nonpathogenic form of E. histolytica as E. dispar in 1993
Epidemiology -​ The third most common parasitic cause of death in the world (after malaria and
schistosomiasis)
Morphology -​ Has three stages - (1) trophozoite, (2) precyst, and (3) cyst (immature and mature)
Life Cycle Host:
-​ Completes its life cycle in single host, i.e. man
Infective form:

MAC
 -​ Mature quadrinucleated cyst
-​ Can resist chlorination, gastric acidity and dessication and can survive in a moist
environment for several weeks

Note: Trophozoites and immature cysts are found in the stool of amoebic patients. They
are not infective forms. They disintegrate in the environment or when exposed to gastric
juice.

Mode of transmission:
-​ Fecal-oral route (most common): infection through contaminated food or water
containing mature quadrinucleated cysts.
-​ Sexual contact: rare transmission, mainly through anogenital or orogenital
contact, particularly among homosexual males in developed countries.
-​ Vector transmission: flies and cockroaches can occasionally carry and spread
cysts from feces to food and water.

(PAGE 27-28)
Development in Man (small intestine):
-​ Excystation:
-​ Asymptomatic cyst passers:
-​ Amoebic dysentery:
-​ Amoebic liver abscess:

Development in Man (large intestine)

-​ Encystation:
Pathogenesis -​ Trophozoite of E. histolytica is the major invasive form and possesses many
virulence factors
Pathogenesis of Intestinal -​ Trophozoites invade the colonic mucosa.
Amoebiasis -​ They cause ulcerative lesions and profuse bloody diarrhea (amoebic dysentery).
-​ Affects males and females equally (1:1 ratio).

Amoebic ulcer:
-​ Classical ulcer is flask-shaped (broad base with narrow neck)
-​ Ulcers may be located in the ileocecal region (most common), sigmoidorectal
region, or throughout the large intestine.
-​ Ulcers are scattered with normal mucosa in between.
Types of ulcers:
-​ Superficial (heals without scar)
-​ Deep (heals with scar).
-​ Sizes range from pinhead to inches.
-​ Shapes can be round to oval.
-​ Margins are ragged and undermined.
-​ Base formed on muscle coat.

Complications of Intestinal Amoebiasis

-​ Fulminant amoebic colitis: generalized necrotic involvement of entire large
intestine
-​ Amoebic appendicitis: results when the infection involves appendix
-​ Intestinal perforation and amoebic peritonitis: occurs when the ulcer progresses
beyond the serosa
-​ Toxic megacolon and intussusception (segment of intestine invaginates into the
adjoining intestinal lumen, causing bowel obstruction)
-​ Perianal skin ulcers: by direct extension of ulcers to perianal skin

-​ Amoeboma (amoebic granuloma): A diffuse pseudotumor like mass of

granulomatous tissue found in rectosigmoid region
-​ Chronic amoebiasis: thickening, fibrosis, stricture formation with scarring and
amoeboma formation.
-​ CNS INVATIONp: SECONDARY

MAC
Pathogenesis of -​ Liver is the most common site (because of the carriage of trophozoites through
Extraintestinal the portal vein) followed by lungs, brain, genitourinary tract and spleen.
Amoebiasis
(PAGE 30)
Amoebic Liver Abscess: (male and female ration 9:1)
-​ Most common affected site is the posteriors-superior surface of the right lobe of
liver. Abscess is usually single or rarely multiple.
Anchovy sauce pus:
-​ Liver abscess pus is thick chocolate brown in color.
-​ Fluid is acidic and pH 5.2 – 6.7

Complications of Amoebic Liver Abscess:

-​ With continuous hepatic necrosis, abscess may grow in various direction of liver
discharging the contents into the neighboring organs
-​ Right sided liver abscess: causing granuloma cutis
-​ Left sided liver abscess: amoebic pericarditis
Clinical Manifestations of Asymptomatic amoebiasis:
Amoebiasis -​ 90% of infected persons are asymptomatic carriers and excrete cysts in their feces
-​ It is confirmed that many of these carriers harbor E. dispar
-​ remaining 10% of people (who are truly infected by pathogenic E. histolytica)
Intestinal amoebiasis:
-​ incubation period varies from one to four weeks by four clinical forms:
-​ Amoebic dysentery: Symptoms include bloody diarrhea with mucus and pus cells,
colicky abdominal pain, fever, prostration, and weight loss. Amoebic dysentery
should be differentiated from bacillary dysentery
-​ Amoebic appendicitis: Presented with acute right lower abdominal pain
-​ Amoeboma: It present as palpable abdominal mass
-​ Fulminant colitis: Presents as intense colicky pain, rectal tenesmus, more than 20
motions/day, fever, nausea, anorexia and hypotension.
Amoebic liver abscess:
-​ Presents with tender hepatomegaly, fever with weight loss, sweating and
weakness, rarely jaundice, and cough
Laboratory Diagnosis of Sample collection:
Intestinal Amoebiasis Stool microscopy:
Stool culture:
Polyxenic culture:
Axenic culture:
Serology:
Molecular diagnosis:

MAC
Laboratory Diagnosis of Microscopy:
Amoebic Liver Abscess -​ Microscopy of liver pus can detect trophozoites (but never cyst) (< 25%
specificity)
-​ It confirms the diagnosis
-​ Stool microscopy is not useful
Stool Culture:
-​ Though it is considered as the gold standard test, the sensitivity is low (<25%)
Antigen Detection:
-​ Lectin antigen can be demonstrated in serum (70% sensitive in late stage, 100%
sensitive when tested before treatment)
-​ liver pus (100% sensitive when tested before treatment) and saliva (70%
sensitive)
Antibody Detection:
-​ - Antibody detection methods for amoebic liver abscess diagnosis include IHA,
IFA, ELISA, CIEP, CFT, SAT, and CIA.
-​ Antibodies can persist after treatment, making it difficult to distinguish between
recent and old infections.
Prevention -​ Avoidance of the ingestion of food and water contaminated with human feces
-​ Treatment of asymptomatic persons who pass E. histolytica cysts in the stool may
help to reduce opportunities for disease transmission

Vaccination:
-​ No effective vaccine for E. histolytica has been licensed for human use yet
-​ Colonization blocking vaccines are currently in
trials The trials target three specific antigens:
-​ SREHP (170 kDa subunit of lectin antigen)
-​ 29 kDa cysteine-rich protein

MAC
NON PATHOGENIC/ COMMENSAL INTESTINAL AMOEBA

ENTAMOEBA ENDOLIMAX IODAMOEBA

NUCLEUS ●​ Spherical nucleus w/ lined ●​ VESICULAR NUCLEUS ●​ Large chromatin-rich

chromatin granules ●​ Large and irregularly-shaped karyosome surrounded
●​ Small karyosome near the karyosome ANCHORED TO by a layer of achromatic
center of the nucleus. THE NUCLEUS by globules and anchored to
●​ Troph only has ONE achromatic fibrils the nuclear membrane
nucleus. by achromatic fibrils

ALL HAVE TROPH, PRECYST, CYST, METACYST TROPH AND RESIDES SA INTESTINE, EXCEPT NI
ENTAMOEBA GINGIVALIS, IT HAS NO CYST STAGE AND RESIDES IN THE MOUTH.

Entamoeba dispar
-​ Morphologically indistinguishable (both cyst and trophozoite) from E. histolytica,
so it may be considered as a subspecies of E. histolytica.
It can be distinguished from E. histolytica by:
-​ Zymodeme study (hexokinase isoenzyme pattern)
-​ Molecular methods, PCR amplifying small subunit rRNA gene)
-​ Detection of lectin antigen in stool
-​ NO RBC inside trophozoites—present only in E. histolytica
-​ Nonpathogenic, usually COLONIZE THE LARGE INTESTINE (10 times more
than E. histolytica) but doesn’t invade intestinal mucosa
-​ Grows well in polyxenic media, usually colonizes in the large intestine
-​ E. dispar doesn’t include antibody production

Entamoeba moshkovskii
-​ is also morphologically indistinguishable from E. histolytica and E. dispar (may
be the third subspecies of E. histolytica) BUT differs biochemically and
genetically.
-​ was first described from Moscow sewage by Tshalaia in 1941
-​ Reported in some areas, such as North America, Italy, South Africa, Bangladesh,
India, Iran, and Australia
-​ PHYSIOLOGICALLY UNIQUE FOR BEING OSMOTOLERANT, CAN GROW IN ROOM
TEMP (25-30°C OPTIMUM) AND ABLE TO SURVIVE AT 0 to 41°C
-​ can be distinguished from E. histolytica by isoenzyme analysis, molecular
methods and detection of lectin antigen
-​ Studies from Bangladesh and India have reported E. moshkovskii as a sole
potential pathogen in patients presenting with gastrointestinal symptoms and/or
dysentery, highlighting the need for further study to investigate the pathogenic
potential of this organism.

Entamoeba hartmanni
-​ also known as small race variant of E. histolytica
-​ morphologically it is similar to E. histolytica but of smaller size (trophozoite is
8– 10 µm and cyst is 6–8 µm)
-​ MATURE CYST IS QUADRINUCLEATED rod-shaped chromatoid material with
rounded or squared ends
-​ nonpathogenic and colonizes the large intestine
-​ life cycle is similar to E. histolytica but does not ingest RBC

MAC
 Entamoeba gingivalis
-​ first parasitic amoeba of humans to be described; recovered from the soft tartar
between the teeth= MOUTH
- VERY ABUNDANT IN CASES OF ORAL DISEASE
- MOT: direct
- Kissing, droplet sprays, sharing utensils
It is unusual in two respects:
1.​ inhabits the mouth rather than in the large intestine
2.​ only trophozoite stage exists; no cystic stage

-​ large food vacuoles containing WBCs (ONLY THE ENTAMOEBA SPP HAVE
WBC)
It is recovered from:
-​ Vaginal secretions of women using intrauterine devices
-​ Oral cavities of patients on radiation therapy and human immunodeficiency virus
(HIV) infection
-​ Patients with pyorrhea alveolaris

Entamoeba coli
-​ is a nonpathogenic amoeba that colonizes the large intestine
-​ cosmopolitan in distribution
-​ it also has three forms – trophozoites, precyst, and cyst
-​ frequently found in the stool samples of healthy individuals

MAC
MAC
 Entamoeba polecki
-​ Non Pathogenic amoeba usually found in the intestine of PIGS AND MONKEYS
-​ Human infection is rare
-​ Trophozoites measures 10–12 µm size AND motility is SLUGGISH like E. coli
-​ Motility non progressive and sluggish
-​ contains one nucleus having central karyosome and fine peripheral chromatin
-​ Cyst is of 5–11 µm size, has one nucleus with features similar to that of trophozoite
-​ It has many chromatoid bodies with threadlike ends (like E. coli) and cytoplasm has
a large non glycogen inclusion mass
-​ Common in areas where pig-to-human and human-to-human exist

Entamoeba chattoni
-​ Morphological identical to E. Poleki
-​ FOUND IN APES AND MONKEYS
-​ ISOENZYMES ANALYSIS IS DONE FOR IDENTIFYING ENTAMOEBA CHATTONI

Endolimax nana
-​ Is a small (nana means small) non pathogenic amoeba
-​ OCCURS IN THE SAME FREQUENCY AS E. COLI
-​ Frequently resides in the large intestine of humans and other animals
-​ Trophozoite measures 8–10 µm in size and shows sluggish motility. THEY ARE
BLUNT W/ HYALINE PSEUDOPODIA
-​ Cyst is 6–8 µm in size and contains one to four nuclei/ QUADRINUCLEATED
-​ Cytoplasm doesn’t have a chromatoid body or glycogen vacuole. HAS FOOD
VACUOLE CONTAIN BACTERIA
-​ Karyosome is eccentric, no peripheral chromatin on nuclear membrane

MAC
 Iodamoeba bütschlii
-​ is also worldwide in distribution though less common than E. coli and E. nana
-​ Trophozoite is 12–15 µm in size
-​ The ectoplasm and endoplasm are not differentiated.
-​ Cyst measures 10–12 µm in size, round to oval and mostly is uninucleated
-​ Bull’s eye appearance or basket nucleus
-​ DOES NOT STAIN THE GLYCOGEN BODY
-​ Cytoplasm of the cyst contains large iodine stained glycogen mass or
acidophilic body (hence named as Iodamoeba)

MAC
FREE-LIVING AMOEBA

-​ These amoebae are small, freely living, widely distributed in soil and water and can cause opportunistic
infections in humans.
-​ Only four genera have an association with human disease. They are:
1.​ Naegleria fowleri is a causative agent of primary amoebic meningoencephalitis (PAM)
2.​ Acanthamoeba species causes granulomatous amoebic encephalitis (GAE) and amoebic keratitis in contact lens
wearers
3.​ Balamuthia mandrillaris causes GAE
4.​ Sappinia diploidea
They differ from intestinal amoeba by:

+ Naturally found freely outside the host in the environment (soil and water)

+ Possesses plenty of mitochondria (intestinal amoeba lack mitochondria)

+ Nuclear membrane is distinct, not lined by peripheral chromatin granules and nucleolus is large, deep stained.
(Intestinal amoeba has a delicate nuclear membrane, small pale stained nucleolus)

+ Cause opportunistic infection affecting central nervous system (CNS)

MAC
 Naegleria fowleri
-​Is a free-living amoeba, typically found in warm fresh water (ponds, lakes, rivers
and hot springs)
-​ Also found in soil, near warm-water discharges of industrial plants and swimming
pools
-​ HEAT-LOVING AMOEBA (thermophilic) thrives in warm water at low oxygen
tension
-​ Cause Primary amebic meningoencephalitis (PAM)
-​ N. australiensis and N. italica, can cause infection in mice
-​ also known as “the brain eating amoeba”
Morphology Trophozoite Cyst
-​ two forms: amoeboid and
flagellated form (both measure
8–15 µm)
Amoeboid form:

-​ Troph encyst due to unfavorable

conditions like food
deprivation, desiccation, cold
temp, etc.
-​ Measures 7–15 µm in size and is
-​ 10-20 µm surrounded by a thick,
-​ Vacuole's appearance is called SMOOTH DOUBLE WALL.
AMEBOSTOME. They are used -​ Nucleus is identical to that found
to engulf RBC AND WBC. in the trophozoite.
-​ Spherical nucleus with big -​ RESTING AND DORMANT FORM
endosome and pulsating can resist in unfavorable conditions
vacuoles such as drying and chlorine
-​ is the ONLY RECOGNIZABLE exposure up to 50 ppm
FORM IN HUMANS -​ Cysts are not found in tissue
-​ possesses lobate pseudopodia (humans) but can be grown in
(called LOBOPODIA) culture.
-​ it is the only replicating form -​ The cyst can withstand
and it divides by binary fission moderate heat ( 45°C), but
-​ INVASIVE, INFECTIVE, FEEDING, die at chlorine levels of2
GROWING, AND REPLICATING ppm and salinity of 0.7%
FORM
Flagellated form:

-​ Amoeboid forms can change

shape when exposed to
different ionic concentrations
(eg. Distilled water).
-​ Show JERKY OR SPINNING
MOTILITY.
-​ When placed in distilled water
at 27–37°C, they quickly
transform into a pear-shaped
biflagellated form.
-​ The new form has TWO
FLAGELLA/ BIFLAGELLATE
FORM at the broader end. - This
transformation occurs within a
few hours.
-​ The organisms exhibit
typical jerky or spinning
MAC
 movement.
-​ When the flagella are lost, they
revert back to amoeboid form
hence THIS PARASITE IS
CLASSIFIED AS
AMOEBOFLAGELLATE.
Life cycle Infective form: AMOEBOID FORM
MOT: Nasal contamination during swimming in fresh hot (still) bodies of water.

CNS INVASION:
-​ Ameboid invade the nasal mucosa →cribriform plate →travels →olfactory nerve →brain
-​ Penetration result in significant necrosis and hemorrhages in nasal mucosa and olfactory
bulbs

Pathogenicity

Clinical features Primary amoebic meningoencephalitis (PAM)

-​ ACUTE suppurative fulminant infection
-​ Usually occurs in HEALTHY CHILDREN OR YOUNG ADULTS swimming in fresh hot
water
-​ Incubation period: 1-2 days to 2 weeks
-​ INITIAL SYMPTOMS:
-​ Change in taste and smell (due to olfactory invasion)
-​ Headache
-​ Anorexia
-​ Nausea
-​ vomiting
-​ High fever
-​ signs of meningeal involvement like stiff neck and a positive Kernig’s sign

-​ SECONDARY SYMPTOMS:
-​ Confusion
-​ Hallucination
-​ Lack of attention
-​ Ataxia
-​ Seizures

-​ MORTALITY RATE IS NEARLY 98%

-​ DEATH OCCURS W/IN 7-14 DAYS AFTER EXPOSURE
Laboratory Diagnosis Cerebrospinal fluid analysis

MICROSCOPY
Direct microscopy
-​ Motile amoeboid troph can be seen in WET MOUNT OF CSF
Phase contrast microscope
-​ yields better result than light microscope
Histopathological staining
-​ Wright or giemsa stain of CSF or BRAIN BIOPSIES
-​ TROPH HAVE SKY-BLUE CYTOPLASM W/ PINK NUCLEUS

MAC
 -​ REFRIGERATION IS NOT RECOMMENDED IF THERE'S DELAY IN EXAMINING THE CSF
-​ If the parasite load is low then CSF can be centrifuged at low speed (150 rpm for 5
minutes). Trophozoites are not damaged, they only lose their pseudopodia
-​ Trophozoites can also be demonstrated by direct fluorescence antibody staining of
centrifuged CSF using monoclonal antibody
Culture
-​ Cultivated on non nutrient agar (Page’s saline and 1.5% agar)
Flagellation test
-​
Isoenzyme Analysis
-​
Molecular Methods

Imaging Methods
-​
Treatment -​ No effective treatment is available for PAM
-​ Amphotericin B has considerable anti-Naegleria effect. Four cases were treated
successfully with amphotericin B.
-​ Other drugs like rifampicin, azithromycin and antifungals like miconazole and
voriconazole are also found to be effective

Acanthamoeba spp.
-​ Is ubiquitous and present worldwide
- A. culbertsoni {formerly, HartmannelLa culbertsoni) is the species most often responsible
for human infection but other species like A. polyphagia, A. castellanii and A. astromyx have also
been reported

DISTRIBUTION -​ WORLD WIDE

-​ 400 CASES REPOTED
Morphology TROPHOZOITE CYST
-​ INFECTIVE STAGE -​ POLYGONAL DOUBLE-WALLED CYST
-​ 20- 50 µ -​ THEY ARE PRESENT IN ALL TYPES OF
-​ HAS SPINE-LIKE PSEUDOPODIA (For ENVIRONMENT
locomotion) (ACANTHOPODIA) -​ INFECTIVE STAGE
-​ Single nucleus w/ centrally located
densely STAINING NUCLEOLUS
-​ REPLICATE BY MITOSIS

MAC
Life cycle
MOT: acquire by inhalation of cyst or trophozoite - CAN ENTER IN VARIOUS WAYS
After inhalation of aerosol or dust containing trophozoites and cysts, the trophozoites reach the
lungs and from there, they invade the central nervous system through the bloodstream, producing
granulomatous encephalitis

Pathogenesis Acanthamoeba keratitis

-​ associated with the use of improperly disinfected soft contact lenses
-​ Immunocompromised increase susceptibility and MAY LEAD TO GAE
-​ SYMPTOMS:
-​ Severe ocular pain
-​ Blurring vision
-​ MAY LEAD TO:
-​ Corneal ulceration
-​ Hypopyon formation from primary amebic infection and bacteria infection
-​ VISION LOSS
Granulomatous Amebic Encephalitis
-​ usually occurs in immunocompromised hosts including the chronically ill and debilitated, and
those on immunosuppressive agents such as chemotherapy and anti-rejection medications
-​ Insidious onset: Incubation period varies from several weeks to months
-​ Chronic course: Lasts for months to years
-​ HAVING AIDS DRAMATICALLY INCREASE GAE
-​ INCUBATION PERIOD: appx. 10 days
-​ Systemic manifestations early in the course include fever, malaise, and anorexia
-​ Neurologic symptoms:
MAC
 -​ INCREASE SLEEPING TIME
-​ Severe headache
-​ Mental status change
-​ Epilepsy
-​ Coma

-​ Neurologic findings depending on the location of the lesions include hemiparesis, blurring of
vision, diplopia, cranial nerve deficits, ataxia, and increased intracranial pressure.
-​
READ BILIZARIO FOR MORE INFO
Laboratory Acanthamoeba keratitis
diagnosis -​ Diagnosed by EPITHELIAL BIOPSY OR CORNEAL SCRAPING (cyst demonstration)
-​ staining patterns on histologic analysis. Amebae have also been isolated from the contact lens
and lens solution of patients
-​ Species-specific identification can be made from culture and molecular analysis through PCR
-​ Rapid diagnosis via corneal scraping by fluorescent microscopy using calcofluor white staining
and IFA Lest (IFAT) procedure
Granulomatous Amebic Encephalitis
-​ Diagnosed by BRAIN BIOPSY, Culture and immunofluorescence microscopy
-​ CEREBROSPINAL FLUID

Treatment Acanthamoeba keratitis

-​ Administration of biguanide or chlorhexidine w/ or w/o diamidine agent.
-​ If SEVERE CASES PENETRATING KERATOPLASTY can be done
Granulomatous Amebic Encephalitis
-​ NO EFFECTIVE AVAILABLE TREATMENT
-​ Multidrug combinations including pentamidine, sulfadiazine, rifampicin and fluconazole are
being used with limited success

MAC
 Balamuthia mandrillaris
-​ is a free-living, heterotrophic amoeba that also causes GAE.
-​ name goes in the honor of the late Professor William Balamuth and it was first discovered in a
pregnant mandrill
Epidemiology -​ distributed in the temperate (cold) regions
Morphology Trophozoite Cyst
-​ is approximately 30 µm, -​ measures 6–30 µm,
-​ irregular with finger like pseudopodia -​ surrounded by a three layered cell wall
-​ OUTER: wrinkled ectocyst
-​ MIDDLE:mesocyst
-​ INNER: thin endocyst
abnormally large Vesicular nucleus.

Life cycle -​ is similar to Acanthamoeba

-​ has trophozoite and cyst form (no flagellated form)
-​ ENTER THE BODY THROUGH RESPIRATORY TRACT

Clinical -​ may enter the body through the respiratory tract or through open wounds
features -​ In CNS, it causes GAE. It also can cause skin lesion.

MAC
Diagnosis Microscopy:
-​ CSF microscopy reveals trophozoites and cysts
Culture:
-​ can be cultured on monkey kidney cell line, HEp2, Vero and diploid macrophage cell line
-​ doesn’t grow on agar plate culture coated with bacteria.

It can be differentiated from Acanthamoeba species by:

-​ Microscopy: Nucleus contains more than one nucleoli and cyst wall is tri layered
-​ IFAT using specific antisera
-​ Culture on cell lines but not in agar plate
-​ PCR targeting mitochondrial small subunit rRNA gene

Sappinia diploidea
-​ newly recognized pathogenic free-living amoeba
-​ found in soil and water
-​ both trophozoite and cyst stages are binucleated
-​ Trophozoite is oval, measures 40–70 µm
-​ Mature cyst is round and measures 15-30 µm
-​ can be cultivated on non-nutrient agar plate coated with bacteria

MAC