Amebiasis

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

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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) brain

cribiform plate

• 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 trophozoite

E. Histolytica immature 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 ofstrips diluted coated stool The immobilized In a positive When a is stop with Monoclonal sample antibodies added E.E. Addition of antibodies bind to any sample, the solution is Hystolitica antibodies substrate TMB hystolitica antigens solution turns added to the (horseradish (tetramethylbenzi present in the sample blue color. blue color, peroxidase the dine) solutionanti-E. turns labeled 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

E. Histolytica test

EIA

Antigen

Fecal

Wampole

E. Histolytica test

EIA

Antigen

Fecal

TechLab

RIDASCREEN Entamoeba

EIA

Antigen

Fecal

ProSpecT E. Histolytica Assay

EIA

Antigen

Fecal

RBiopharm GmbH Alexon

Entamoeba Histolytica

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 • • •

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• •

Test to detect significantly raised levels of antiE.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 

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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.