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AMEBIC INFECTIONS

Introduction:

Amebas
 exist as either obligate parasites that thrive
only when in a host, or as free-living amebas
that exist primarily in the environment and
only occasionally cause human infection.
Two Forms:

a. Trophozoites- motile, actively feeding


 -the motility of the trophozoite is achieved
by extension of pseudopodia. Extended
pseudopodia are also used to engulf matter
that serves as food.
 b. Cyst- dormant
 - are produced when the environmental
conditions are unfavorable for survival of the
trophozoite. In the process of converting to
the cyst form, the ameba discharges any food
that it has ingested and becomes
transformed to a round shape with a tough
membrane.
Clinical
manifestations/pathology
Acathamoeba

 free-living amebas found throughout the


environment, including tap water, bottled
mineral water, chlorinated swimming pools,
sea water and soil.
Habitat in man:
Respiratory tract eyes brain
Two forms:
a. trophozoite pseudopods – spine-like/thorny
appearance
b. cyst – double walled, wrinkled, ruffled
Causes:
• Sinusitis
• Dermatitis
• Pneumonitis
• Amebic keratitis

-Local tissue damage, multinucleated giant


cells, and lots of activated mononuclear
phagocytes characterize granulomas. With the
exception of amebic keratitis, genetic or
acquired immunosuppression is the main risk
factor for disease caused by Acanthamoeba
spp.
Primary sites for disseminated infection:
• Lungs
• Abraded skin

Inhalation of the cyst from dust aerosol


lungs circulation brain symptoms
(nausea, vomiting, seizures, altered mental
state,infammation)

• These symptoms signal the beginning of slow,


progressive and invariably fatal
granulomatous amebic encephalitis.
Amebic keratitis
• is a painful corneal ulceration that, if left
untreated, can result in a vascular scar over
the cornea, resulting in impaired vision or lost
of the eye.
• Occurs in immunocompetent individuals who
have used nonsterile solutions such as tap
water or home made saline to wash their
contact lenses.
• Infection comes from direct contact of the
ameba with abraded corneal epithelium as
may occur from wearing contact lenses.
• Photophobia – extreme sensitivity to light,
blurred vision and pain.
Acanthamoeba keratitis

Acanthamoeba in brain

Acanthamoeba cysts
Naegleria fowleri
a.k.a : “the brain-eating amoeba”
• Causes primary amebic meningoencephalitis
Found in:
soil, warm fresh water, shallow lakes and
ponds
Habitat in man:
meninges of the brain
IS/DS:
trophozoite
MOT:
nasal entry
Primary amebic meningoencephalitis (PAM)

• “Roman bathspa disease”


• Is a syndrome affecting the CNS, characterized by
changes in olfactory perception , followed by
vomiting, nausea, fever, headache, and the rapid
onset of coma and death in 2 weeks.
• Fatality rate: >95%
• Occurs primarily in children and in young adults

 Warm water infected with N. fowleri enters the


nasal cavity(inhalation) cribiform plate
brain

• Infection w/o exposure to warm contaminated


water most likely represent inhalation of dust that
is ladened with N.fowleri cyst.
Symptoms:
• Fever
• Severe headache
• Nausea
• Vomiting
• Stiff neck
• Disturbances in taste, smell and hearing
• Seizures
• hallucinations
Causes:
• Hemmorhage
• Necrosis of the olfactory bulbs and cerebral
cortex

- The brain becomes swollen as a result of


edema
- Patient lapses into a coma and death
invariably follows

- During autopsy, the amebic trophozoites can


be isolated from the brain. Some of these
contain RBCs and brain tissues that have been
engulfed by the ameba.
Immunity
Cellular response
 Non-pathogenic strains of N. fowleri can be
taken up by antigen-presenting cells after
they have been tagged by antibodies
 APC lymph nodes present the ag
to CD8+ cytotoxic T cells and CD4+ helper T
cells
 B cells are also activated and they produce
more ag specific antibodies.
Hmoral response:
 When N. fowleri invades a host, it can be
phagocytosized by the APCs.
 APCs then go to the lymph nodes and
presents the angtigen to CD4+ helper T cells.
 Activated T cells activate B cells which
differentiate into memory B cells and plasma
cells then secrete antibodies which binds to
N.fowleri.
Entamoeba histolytica
 Most pathogenic, disease causing, intestinal
ameba
 Tissue invading parasite
Causes:
amoebiasis, amoebic dysentery or amoebic
liver abscess
MOT:
fecal-oral route
Habitat:
GIT (small intestine, large intestine, rectum)
Vector:
Musca domestica – common household fly
Naegleria Fowleri
Trophozoite
Acute intestinal form:
 Abdominal cramping
 Nausea
 Severe diarrhea (mucus&blood are
+)
Complications:
 Dissemination to the liver
 Liver abscess – fatal if untreated
E. Histolytica immature
E. Histolytica trophozoite cyst

E. Histolytica mature cyst


Immunology

 Three E. histolytica structural proteins have


been identified as antigenic and capable of
eliciting antibody responses:
- 170 kD subunit of the lectin protein
(adhesin)
- serine rich Entamoeba histolytica protein
(SREHP)
-2 kD cysteine rich protein
Humoral Immunity:
Detected in patients with amebic colitis:
 Serum anti-E.histolytica IgA & IgM antibodies
 Fecal secretory IgA anti-amebic antibodies
Detected in saliva of children with intestinal
amebic infection:
 Anti-e. histolytica secretory IgA antibodies

- however, whether the presence of these


antibodies is protective for subsequent
infection is not known.
Cellular Immunity:
 Serum from patients with amebic hepatic
abscess sverely inhibits antigen-induced
proliferation of T cells and production of type
1 cytokines, in vitro.
 Type 1 cytokines enhance the cytotoxic
activity of phagocytes, this form of
suppression represents an evasive strategy
that has evolved to counter host responses.
 Attempts to identify the molecules that inhibit
T cell function have shown that the
suppressive activity was not Ab mediated.
Antigen Detection

-The most rapid, reliable, and technically simple


effective test is the enzyme immunoassay

-Detects antigen in an aqueous extract of human fecal


specimens
Immunization Progress:
 E. histolytica vaccines are still in pre-clinical
phase.
Human Fecal Specimen (antigen)

Antibodies to E. Hystolitica

Monoclonal antibody
(Horseradish peroxidase
labeled anti-E. Hystolitica
antibody)

Substrate TMB
(tetramethylbenzidine)
Microwell
Second
Aliquot
The ofstrips
diluted
immobilized coated
stool
In a
Whenpositive
a is
stop
with
Monoclonal
sample
antibodies
Addition of
antibodies added
bind to E.E.
any
sample, the
solution is
Hystolitica
antibodies
substrate
hystolitica TMB
antigens
solution
added toturns
the
(horseradish
(tetramethylbenzi
present in the sample
blue color.
peroxidase the
blue
dine)
color,
solutionanti-E.
labeled turns
yellow.
Hystolitica
antibody) is
added
ELISA for Detecting
Antibody
• The technique is widely thought to be sufficient for clinical
purposes (particularly in diagnosing ALA patients), since the
value of specific antibodies detected in symptomatic
patients is thought to be high
• Serum anti-lectin immunoglobulin G (IgG) antibodies could be
present within 1 week after the onset of symptoms of
patients with amebic colitis and ALA
Test Principle Detect Sample Company
Entamoeba Histolytica
E. Histolytica test EIA Antigen Fecal Wampole
E. Histolytica test EIA Antigen Fecal TechLab
RIDASCREEN Entamoeba EIA Antigen Fecal R-
Biopharm
ProSpecT E. Histolytica EIA Antigen Fecal GmbH
Alexon
Assay Trend
Indirect Hemagglutination test for Amoebiasis

• Detects antibody specific for E histolytica.


The antigen used in IHA consists of a crude
extract of axenically cultured organisms

• The test is highly specific (³96%)


in
extra intestinal amoebiasis,
especially ALA
1:128 titer is considered
indicative of invasive amoebiasis.
• The sensitivity of IHA was 72.4%
in patients with ALA 1 and 2
weeks after the onset of
symptoms, but it was 86.9% at
the end of week 3.
Antigen--prepared from axenically
grown E. histolytica strain HK-9

Sheep RBC or Human


RBC type O

Patient’s sample
Cellulose Acetate
Precipitin (CAP) Test
• Test to detect significantly raised levels of anti-
E.hystolitica antibodies in serum.
• Highly specific for invasive amoebiasis.
• This test relies on the surface diffusion of specific globulins
and soluble antigen to produce a line of precipitation where
they meet which can be visualized by staining.
• A Cellulose Acetate Precipitin test (CAP) will be performed
if the IFAT is positive. This test is less sensitive than the
IFAT.
• A positive is confirmatory evidence of active infection.
• A negative CAP in the presence of a positive may suggest
early infection
Indirect Flourescencent Antibody
Test

 Antigens used are E.histolytica strains HK-9


and HM-1 in Diamond’s medium.
 Ag were dosed in wells made on glass slide
and overlaid with serial dilutions of the
patient’s serum.
 IgG gamma chain-specific flourescein
isothiocyanate conjugate were added.
 Positive reaction indicating the presence of ab
would exhibit luminescence or a bright green
color under an ultraviolet microscope.
 Titer is read at the highest serum dilution
where definite flourescence was bserved after
incubation for 1 hour.
 Titer of 1:64 is considered positive
Indirect
immunofluorescence for
Acanthamoeba and N.
Fowleri

Acanthamoeba N. Fowleri
Molecular Diagnosis

 Molecular diagnostic assays have been


developed to aid in the diagnosis of E.
Histolytica infections.
 These assays are based on the amplification
of a segment of E. histolytica ribosomal RNA
(or a segment of DNA) using PCR on a clinical
stool specimen.