Parasitology-lec 10 Entamoeba

PARASITOLOGY LECTURE 10 – Intestinal Amebae, Commensal Amebae and Free-Lining Pathogenic Amebae Notes from Rivera,PT, Rivera,WL and Solon,JAA USTMED ’07 Sec C – AsM

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INTESTINAL AMEBAE ENTAMOEBA HISTOLYTICA

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Classification o subphylum Sarcodina o superclass Rhizopoda o class Lobosea o order Amoebida o family Entamoebidae o genus Entamoeba

DISEASE

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cause invasive intestinal and extraintestinal disease

life cycle is simple and no intermediate hosts involved extracellularly located and do not undergo antigenic variation Cyst o Quadrinucleate o Resistant to gastic acidity and dessication o Can survive in a moist environment for several weeks o Infection occurs when cysts are ingested from fecally-contaminated material o Modes of transmission  Fecal-oral route  Direct colonic inoculation through contaminated enema equipment o Excystation occurs in the small or large bowel a. nuclear fission b. cytoplasmic division (forms 8 trophozoites)

immature cysts

MORPHOLOGY

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a pseudopod-forming nonflagellated protozoan parasite the most invasive in the Entamoeba family (which includes E. dispar, E. hartmanni, E.polecki, E. coli and E. gingivalis) only member of the family to cause colitis and liver abscess a eukaryotic organism cellular features o lack organelles that resemble mitochondria o no ER o no Golgi apparatus o cell surface and secreted proteins contain signal sequences o Ribosomes form aggregated crystalline arrays in the cytoplasm in trophozoite biochemical characteristics o lack glutathione metabolism o uses pyrophosphate instead of ATP at several steps in glycolysis o inability to synthesize purine nucleotides de novo o glucose is actively transported into cytoplasm o end products of metab are EtOH and CO2 (acetate in aerobic conditions)

mature cysts

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LIFE CYCLE

Trophozoites o Highly motile o Possess pseudopodia o Have the ability to colonize and/or invade the large bowel (cysts are never found w/in invaded tissues) o Multiply by binary fission o Encyst a. produces uninucleate cysts b. undergo 2 successive nuclear divisions (forms quadrinucleate cysts)

PATHOGENESIS AND CLINICAL MANIESTATION

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2 stages 1. Infective cyst 2. invasive trophozoite form

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humans are the only known hosts

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most cases present as asymptomatic infections with cysts being passed out in the stools (cyst carrier state) the non-pathogenic E. dispar has a higher prevalence than E. histolytica most E. histolytica infections are asymptomatic in endemic communities Amebic colitis o Gradual onset of abdominal pain and diarrhea w/ or w/o blood and mucus in stools o Fever occurs only in 1/3 of patients o Intermittent diarrhea alternating with

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constipation Children may develop fulminant colitis  Severe bloody diarrhea  Fever

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Abdominal pain

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colitis

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Ameboma o Occurs in less than 1% of intestinal infections o Mass-like lesion with abdominal pain and history of dysentery o Can be mistaken for carcinoma

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Amebic liver abscess (ALA) o Most common extra-intestinal form of amebiasis o Cardinal manifestations (most frequent complaints in acute cases {<2 weeks duration})  Fever  Right Upper Quadrant pain (RUQ) – local or referred to the right shoulder shoulder o Liver is tender o Hepatomegaly is present in 50% of cases o Chronic disease (>2 weeks duration)  found in older patients  Wasting disease  Weight loss rather than fever o 72% of daily stool cultures harbored trophozoites even in asymptomatic infections o mortality uncomplicated <1%

this initial condition Liver involvement occur through direct extension from the intestinal ulcer In the liver, trophozoites lyse both inflammatory and liver cells Abscess becomes filled with necrotic proteinaceious debris (anchovy sauce-like aspirate) Trophozoites are found at the edge of the abscess

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Aspirate = odorless and bacteriologically (secondary bacterial invasion may occur)

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Complications o Amebic Colitis:  Perforation and Secondary Bacterial peritonitis – most serious complication o ALA  Rupture into the pericardium, rupture into the pleura and super infection – most serious

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Intraperitoneal rupture – second most common complication (not as serious because ALA is sterile) Secondary amebic meningoencephalitis – considered in cases with abnormal mental status Renal involvement caused by extension of ALA ore retroperitoneal colonic perforation (rare) Genital involvement – caused by fistulae from ALA and colitis or infection by sexual transmission

Immunity o

Natural or innate immunity  In the intestines – involves mucin inhibition of amebic attachment to underlysing mucosal cells  Systemic circulation – mechanism is of complement-mediated killing of trophozoites

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Acquired immunity  Involves cell-mediated and humoral responses

 PATHOLOGY

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E. histolytica named because of its ability to lyse human tissues Invasive process initiated when trophozoite stage is able to penetrate mucus layer covering the colonic epithelium Invasion facilitated by expression of virulence factors 1. Gal/Gal NAc lectin – mediates adherence to host cells 2. Amebapores – form pores in host cell membranes 3. cystein proteinases – cytopathic for host tissues In amebic invasion, Trophozoites… o Cause thinning of mucin layer action of o Shortening of villi cystein o Breakdown of extracellular matrix proteases o Attach to mucosal cells facilitated by lectin o Lyse cells amebapores o Stimiulate release of IL8 which attracts and activates neutrophils o Trophozoites erode the lamina propria and extend laterally producing characteristic flaskshaped ulcer

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responses

Activated T-cells kills E. histolytica i. by lysing trophozoites (by contact) ii. producing cytokines w/c activate macrophages and effector cells iii. providing helper effect for Bcell Ab production

Amebic modulation of host immune responses  Infected subjects have been shown to be in a state of immunosuppresion during the acute stage i. T cell hyporesponsiveness ii. Depressed DTH iii. Macrophage suppression

DIFFERENTIAL DIAGNOSIS

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acute amebic colitis should be differentiated from bacillary dysentery of the ff. etiology: Shigella, Salmonella, Campylobacter, Yersinia, Entero-invasive E. coli) o Fever and significantly elevated leukocyte count are less common in amebic colitis

Comparison of bacillary and Amebic dysentery

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Flask-shape ulcer = small defect in the mucosa and larger area of necrosis in the submucosa and muscularis layers surrounded by normal epithelium Most common sites of amebic ulcer are cecum, ascending colon and sigmoid o o

From the primary site in the colon, trophozoites reach liver through portal vein Causes periportal inflammation  Amebic hepatitis – postulated for

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Baciliary Dysentery Maybe epidemic Acute onset Prodromal fever and malaise common Vomiting common Patient prostrate Watery, bloody diarrhea Odorless stool

Amebic Dysentery Seldom epidemic Gradual onset No prodromal features No vomiting Patient usually ambulant Bloody diarrhea Fishy odor stool

Stool microscopy: numerous bacilli, pus cells, macrophages, red cells, no Charcot-Leyden crystals Abdominal cramps common and severe Tenesmus common Natural history: spontaneous recovery in a few days, weeks or more; no relapse •

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Stool microscopy: few bacilli, red cells, trophozoites with ingested RBCs, Charcot-Leyden crystals Mild abdominal cramps Tenesmus uncommon Natural history; lasts for weeks; dysentery returns after remission; infection persists for years

Differentiate amebic colitis from inflammatory bowel disease o Amebic colitis should be ruled out before steroid therapy is started because of risk of developing toxic megacolon ALA vs. pyogenic liver abscess, TB of the liver and hepatic carcinoma Genital amebiasis vs. carcinoma, TB, chancroid and lymphogranuloma venereum

TREATMENT AND PROGNOSIS

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DIAGNOSIS

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Microscopy standard method of diagnosis of trophozoites and cysts in stool specimens

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for detection of trophozoites o fresh stool sample examined w/in 30 minutes from defecation use DFS with saline solution able to observe trophozoite motility o unidirectional movement is characteristic use saline and methyline blue o Entamoeba species will stain blue (differentiate from WBCs) Use saline and iodine o Nucleus and karyosome can be observed (differentiate E. histolytica from nonpathogenic E. hartmanni, E. coli, Endolimax nana) Detection of E. histolytica trophozoite w/ ingested RBCs is diagnostic of amebiasis. Charcot-Leyden crystals can also be seen in stool

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concentration methods such as Formalin Ether Conc Test (FECT) and Merthiolate Iodine Formalin Conc. Test (MIFC) are more sensitive than DFS for detection of cysts morphologic structures observed: 1. size of cyst 2. number of nuclei 3. location and appearance of karyosome 4. appearance of chromatoid bodies 5. presence of cytolasmic structures such as glycogen vacuole Species Identification of E. histolytica and E. dispar is not possible for microscopy o Done by PCR, ELISA and isoenzyme analysis

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Serology Detection of antibodies in the serum is still the key diagnosis of ALA In ALA, microscopic detection cannot be done because aspiration is an invasive procedure and trophozoites are missed because they are found at the periphery of the abscess Serological tests: Indirect hemagglutination (IHAT), counter immunoelectrophoresis (CIE), agar gel diffusion (AGD), indirect fluorescent antibody test (IFAT) and ELISA o IHAT can detect antibodies of past infection, even as long as 10 years Abs demonstrated in asymptomatic infections so serology can be used to monitor cyst carriers

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EPIDEMIOLOGY

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infections occur worldwide but more prevalent in tropics worldwide cases 50 million, 100,000 of which end fatally second to malaria in terms of mortality caused by protozoan parasites humans are major reservoirs of infection Mexico, Indonesia, Africa, Central and South America, Philippines Risk factors: treatment with corticosteroids, malignancy and malnutrition

PREVENTION AND CONTROL

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Treatment has 2 objections 1. cure invasive disease at both intestinal and extraintestinal sites 2. eliminate the passage of cysts from intestinal lumen Metronidazole is drug of choice o Tinidazole and Secnidazole are also effective o Diloxanide furoate drug of choice for asymptomatic cyst passers. Percutaneous drainage of live abscess o For patients who do not respond to metronidazole o For prompt symptomatic relief of severe pain o For those with left lobe abscess that may rupture into the pericardium (and large and multiple abscess with danger of rupture)

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improve environmental sanitation and sanitary disposal of human feces, safe drinking water, safe food proper use of latrines and proper hygiene such as washing hands Vaccines are cost-effective and a potent strategy for amebiasis prevention

COMMENSAL AMEBAE

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the presence of commensal amebae in stool is significant because: 1. may be mistaken for E. histolytica 2. indication of fecal contamination of food or water

MORPHOLOGY

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Radiographic Ultrasound, CT scan and MRI – non-invasive and sensitive methods in early detection of ALA

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differentiated from pathogenic E. histolytica the three genera of intestinal amebae can be differentiated through morphological features of their nuclei Entamoeba – o spherical nucleus o distinct nuclear membrane lined w/ chromatin granules o small karyosome near the center of the nucleus o trophozoites – usually only have one nucleus Endolimax – o vesicular nucleus o large, irregularly-shaped karyosome anchored

to nucleus by achromatic fibrils 3.

Iodamoeba – o large chromatin-rich karyosome surrounded by a lyer of achromatic globules anchored to nuclear membrane by achromatic fibrils

LIFE CYCLE

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all species have the following stages 1. Trophozoite 2. Precyst 3. Cyst 4. Metacystic trophozoite o exception of E. gingivalis w/c has no cyst stage and does not inhabit the intestines cysts pass through the acidic stomach unscathed, protected by cyst walls excystion occurs in the alkaline environment of lower small intestines Metacystic trophozoites colonize large intestines and live on mucus coat covering intestinal mucosa Amebae are non-invasive and do not cause disease Reproduction by binary fission of trophozoites Encystion occurs as amebae pass through lower colon

ENDOLIMAX NANA

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small size of 6 to 15 um sluggish movement

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characteristic Endolimax nucleus (w/ large irregular karyosome) cysts are quadrinucleate when mature

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cyst

trophozoite

ENTAMOEBA HARTMANNI

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similar to E. histolytica except that it is much smaller and does not ingest RBCs more sluggish movement mature cyst are quadrinucleated and have a coarse cytoplasm immature cysts have chromatoidal bars (short with tapered ends, or thin and bar-like)

IODAMOEBA BüTSCHLII

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cyst

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trophozoite is usually 9-14 um long characteristic large vesicular nucleus w/ large endosome surrounded by achromatic granules no peripheral chromatin granules on nuclear membrane cysts is uninucleated w/ large glycogen body w/c stains deeply w/ iodine

cyst trophozoite

trophozoite

ENTAMOEBA COLI

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cosmopolitan distribution harmless inhabitant of colon typical Entamoeba nucleus Trophozoite can be diff. from E. histolytica by features: o More vacuolated or granular ER w/ bacteria and debris but no RBCs o Narrower, less differentiated ectoplasm o Broader, blunter pseudopodia o More sluggish undirected movements o Thicker, irregular peripheral chromatin w/ large eccentric karyosomes in the nucleus Cysts of E. coli vs. E. histolytica o Larger size o Greater number of nuclei (8 vs 4 in E. histolytica) o More granular cytoplasm o Splinter-like chromatoidal bodies

cyst

DIAGNOSIS

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stool examination liquid stools will show trophozoites formed stools will show cysts DFS to demonstrate trophozoites Formalin-ether concentration technique to differentiate species

TREATMENT

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not necessary because does not cause disease

EPIDEMIOLOGY

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human infection by ingestion of viable cysts in food or water 21% of Filipinos infected with E.coli, 9% - Endolimax nana, 1% Iodamoeba butschlii

PREVENTION AND CONTROL trohpozoites

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The usual…

FREE-LIVING PATHOGENIC AMEBAE LIFE CYCLE

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 Chills  Fatigue  Weight losss o Common  Headache  Confusion  Somnolence  Coma  Hallucinations  Seizures o Neurologic symptoms  Focal hemiparesis  Cranial nerve palsies  Visual disturbances  Ataxia o Increased intracranial pressure can cause papilledema o Skin lesions are an important diagnostic feature of the infection Incubation period is about 10 days o w/ subacute and chronic clinical course of infection that lasts for several weeks o clinical manifestations  mental abnormalities  meningism  localized neurological signs  coma cerebral hemispheres o edematous o soft with hemorrhages and abscesses o most affected areas are posterior fossa, diencephalons, thalamus and brainstem o leptomeninges are opaque w/ purulent exudates

ACANTHAMOEBA

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route of invasion and penetration into the CNS via the circulatory system primary sites of infection are skin or lungs

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DISEASE

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causes granulomatous amebic encephalitis (GAE)

MORPHOLOGY

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Trophozoites o trophozoites exhibit a single and large nucleus with a centrally-located, densely staining nucleolus, a large endosome, finely granulated cytoplasm and a large contractile vacuole o exhibit small, spiny filaments for locomotion known as acanthapodia o sluggish movement w/ polydirectional movement Cysts o Double-walled w/ outer wrinkeled wall and an inner polygonally-shaped wall o Pores or ostioles are seen at the point of contact bet the two walls Presence of naturally-occuring bacterial endosymbionts in Acanthamoeaba sp.

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a small free-living ameba characterized by an active trophozoite stage and a dormant cyst stage. Sluggishly motile trophozoites Feed on gram negative bacteria, blue-green algae, or yeasts Reproduce by binary fission Encysts if the environment is not favorable A ubiquitous organism

PATHOGENESIS AND CLINICAL MANIFESTATION

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pain  hypopyon  loss of vision often confused w/ fungal or herpetic keratitis

Amebic keratitis

LIFE CYCLE

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also an ocular surface pathogen that causes amebic keratitis o associated with the use of soft contact lenses o viable trophozoites can adhere to lenses if not properly cleaned and disinfected o signs and symptoms  corneal ulceration  progressive corneal infiltration and clouding  iritis  scleritis  s eve re

causes GAE o occur in chronically ill and debilitated individuals o with impaired immune defense mechanisms o under immunosuppressive therapy Signs and symptoms of GAE are related to destructive encephalopathy and associated w/ meningeal irritation o Non-specific constitutional manifestations  Fever

DIAGNOSIS

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GAE (A. encephalitis) o Made only after death in majority of cases o Failure to diagnose is the result of a variety of factors  Lack of initial suspicion  Inaccurate early clinical diagnosis  Rapid progression of illness o Has high incidence in AIDS patients w/ low CD4+ T-lymphocytes counts, especially if associated w/ skin lesions and/or sinusitis o

specific diagnosis

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demonstration of trophozoites or cysts in tissues by microscopy can be isolated from CSF and cultured

A. keratitis o Epithelial biopsy for histologic analysis o Isolation of organisms from lens of contact lens weares o Etiologic agents:  A. castellani  A. culbertsoni  A. hutchetti  A. polyphaga  A. rhysoides

TREATMENT AND MANAGEMENT

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w/ appearance of cerebral manifestation, A. encephalitis has a fatal outcome in 3-40 days o some patients respond to treatment with:  5-fluorocytosine  ketoconazole  itraconazole  pentamidine  amphotericin B A. keratitis treatment achieved w/ multidrug treatment if started early o Clotrimazole + o Pentamidine, o Isethionate, o Neosporin o – avoidance of corticosteroids o surgery for A. keratitis

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Trophozoite in spinal fluid

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Acanthamoeba species have been isolated from all kinds of water (sea, fresh, mineral,etc), air, sewage, soil, compost, vegetables, mushrooms, fish, reptiles, birds, mammals in humans, isolated in nasal cavity, throat and intestines as well as cerebral tissue, lung tissue, skin wounds and cornea encephalitis in US Keratitis in Japan, Korea, South America, Germany

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MORPHOLOGY

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Trophozoite easily recognizable under phase contrast microscope o Characteristic lobose monopseudopodium o Very prominent nucleus w/ centrally-located nucleolus o Forms a pair of flagella originating from the tip of a pear-shaped cell body o Transforms into a biflagellated organism Flagellated organism

diagnosis of PAM – based on presence of trophozoites in brain and CSF Naegleria trophozoites can be identified by presence of blunt, lobose pseudopodia and directional motility

TREATMENT

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boiling water is the best possible way of killing trophozoites and cysts regular disinfection of contact lenses free-living amebo-flagellate can exists as an ameba (trophozoite form) and as a flagellate (swimming form) Naegleria gruberi most commonly studied nonpathogenic species Pathogenic species, N. fowleri, causes fatal meningoencephalitis in humans and laboratory animals such as mice o Causes degenerative or cytopathic effects in cell cultures Non-pathogenic can be distinguished from pathogenic by a combinations of cell morphology, culture medium preference, temperature tolerance, lectin sensitivity, isozyme pattern, DNA restriction patterns, mouse pathogenicity and serology Locally occurring species named N. philippinensis Mode of transmission – oral or intranasal routes while swimming in contaminated pools, lakes and rivers

causes gastritis and diarrhea causes primary amebic meningoencephalitis (PAM) – rare disease that leads to inflammation of the brain and destruction of brain tissue N. fowleri able to survie in elevated temperatures (46oC) and 0.5 ug/ml of hyperchlorinated water PAM is characterized by fever, headache, vomiting, signs of meningeal irritation and encephalitis with rapid progression to coma and death o CSF findings of pleocytosis o High percentage of polymorphonuclear cells o Hypoglycorrhachia o Eleveated protein leves

DIAGNOSIS

NAEGLERIA

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trophozoite stage that can transform reversibly into non-reproductive flagellate or a resistant cyst transformation can take place w/in a period of 2-3 hours or up to 3-4 days

PATHOGENESIS AND CLINICAL MANIFESTATION

PREVENTION AND CONTROL

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Trophozoite in CSF

LIFE CYCLE

EPIDEMIOLOGY

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Allows it to move towards a food souce more rapidly

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Amphotericin B – drug of choice for treating PAM o Induces changes in nucleus and mitochondria o Increases proliferation of both rough and smooth ER o Decreases number of food vacuoles o Increases formation of autophagic vacuoles o Inhibiting pseudopod formation o Induces blebbing of the ameba plasma membrane N. fowleri o tolerant between 65o - 100o C o inhibted by 0.2 NaCl and KCl o CaCl2 stimulates encystment o Drying is lethal to trophozoites o Cysts remain viable if rehydrated w/in 23 months o Cysts are non viable if lyophilized

EPIDEMIOLOGY

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all types of water soil is preferred habitat

PREVENTION AND CONTROL

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avoid diving into and swimming gin warm and stagnant freshwater pools, discharge pools, unchlorinated poorly maintained poos or mud-lined lakes and ponds

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