PATHOGENIC FREE LIVING AMOEBA

INTRODUCTION

• Among the many genera of free-living amoebae that exist in nature, members of only four genera have an association with human disease: Acanthamoeba spp. Balamuthia mandrillaris, Naegleria fowleri and Sappinia pedata.

• They are aerobic unlike other amoebae which are anaerobic

Amphizoic amoebae

• They have also been called amphizoic amoebae because these amoebae have the ability to exist as free-living organisms in nature and only occasionally invade a host and live as parasites within host tissue.

Naegleria fowleri

• Naegleria fowleri, commonly found in warm freshwater (like lakes, rivers, and hot springs) and soil, is the only species of Naegleria known to infect people.

• Naegleria fowleri is an amoeboflagellate, as it has a transitory, pear-shaped flagellate stage along with amoeboid trophozoite and resistant cyst stages in its life cycle

MORPHOLOGY

• N.fowleri exists in three forms

- trophozoite or amoeboid form

- flagellate form

- cyst or resting form

Trophozoite

• Found on surface of vegetation and mud.

• The trophozoite moves rapidly by producing rounded pseudopodia(lobopodia) .

• Size → 6-15µm in diameter.

• Slug shaped

• Observed in the CSF and in tissue of brain .

Flagellate form

• Pear shaped cell with 2 flagella

• Found in surface layer of the water

• Rapidly motile

• Not found in CSF or brain

Cyst form

• They are uninucleated and possess double cyst wall.

• Found on the surface of vegetation and mud.

• Not found in CSF or in brain.

Cultivation

• The amoeba grows well on monolayers of E6 and HLF cell cultures.

• N. fowleri can be grown in a cell-free axenic medium.

• Proteose peptone glucose medium

Life Cycle• Naegleria fowleri has three stages, cysts ,

trophozoites , and flagellated forms , in its life cycle.

• The soil amoeba gets transformed from trophozoite form to flagellate form in the water.

• Trophozoites can turn into temporary non-feeding flagellated forms which usually revert back to the trophozoite stage.

• The trophozoites multiply by binary fission.

Life Cycle

• Trophozoites encyst under unfavorable condition and excyst under favorable condition.

• Trophozoites infect humans or animals by penetrating the nasal mucosa and migrating to the brain via the olfactory nerves causing primary amoebic meningoencephalitis (PAM).

• N. fowleri trophozoites are found in cerebrospinal fluid (CSF) and tissue, while flagellated forms are occasionally found in CSF.

Primary amoebic meningoencephalitis (PAM)

• Naegleria fowleri causes an acute, fulminating hemorrhagic meningoencephalitis principally in healthy children and young adults with a history of recent exposure to warm fresh water.

• The striking feature of PAM is the rapid onset of symptoms following exposure.

• The disease progresses rapidly, and, without prompt diagnosis and intervention, death usually occurs within a week or less

Clinical manifestations

• The time from initial contact(swimming, diving, water skiing, or simply immersing head in water) to onset of illness is usually 5–7 days, and may even be as short at 24 h.

• The earliest symptoms are sudden onset of bifrontal or bitemporal headaches, high fever,nuchal rigidity, followed by nausea, vomiting, irritability and restlessness.

• Photophobia may occur late in the clinical course, followed by neurological abnormalities, including lethargy, seizures, confusion, coma, diplopia or bizarre behavior, leading to death within a week.

Diagnosis• A wet-mount of the CSF -

for the presence of actively moving trophozoites.

• Smears of CSF should be stained with Giemsa or Wright stains to identify the trophozoite.

• Cultivation• Fluorescent antibody

staining of CSF• PCR

Treatment• Few patients have survived PAM.• Large dose of antifungal agent amphotericin-B

(1 mg/Kg/day I.V. for several days) or ketoconazole (800 mg daily orally for one month).

• Azithromycin, a macrolide antimicrobial, has been shown to be effective against Naegleria both in vitro and in vivo

Prevention and control• Chlorination of heavily used

swimming pools, especially during summer months.

• In high-risk areas, monitoring of recreational waters for N. fowleri amoebae should be considered by local public health authorities and appropriate warnings posted, particularly during the hot summer months.

Acanthamoeba• Acanthamoeba is a microscopic, free-living amoeba

that can cause rare, but severe infections of the eye, skin, and central nervous system.

• Several species of Acanthamoeba, including A. culbertsoni, A. polyphaga, A. castellanii, A. astronyxis, A. hatchetti, A. rhysodes, A. divionensis, A. lugdunensis, and A. lenticulata are implicated in human disease.

• The important species is A.culbertsoni

• Acanthamoeba spp. have been found in soil; fresh, brackish, and sea water; sewage; swimming pools; contact lens equipment; medicinal pools; dental treatment units; dialysis machines; heating, ventilating, and air conditioning systems; mammalian cell cultures; vegetables; human nostrils and throats; and human and animal brain, skin, and lung tissues.

Morphology• There are two morphological forms (a)Trophozoite -A trophozoite is 20-50µm in size -Rough exterior with several spine

like projections(acanthopoda). (b)Cyst -Spherical and 15µm in diameter.• Both forms can be the source of infection

Life Cycle• Acanthamoeba has only two stages, cysts and

trophozoites , in its life cycle. No flagellated stage exists as part of the life cycle.

• The trophozoites replicate by mitosis.• When Acanthamoeba spp. enters the eye it can cause

severe keratitis in otherwise healthy individuals, particularly contact lens users .

• When it enters the respiratory system or through the skin, it can invade the central nervous system by hematogenous dissemination causing granulomatous amebic encephalitis (GAE) or disseminated disease , or skin lesions in individuals with compromised immune systems

Pathogenicity and clinical features

• Granulomatous Amebic Encephalitis (GAE) and disseminated infection primarily affect people with compromised immune systems.

• Commonly seen in immunocompromised patients, including those with neoplasia, systemic lupus erythematosus, human immunodeficiency virus and tuberculosis

• Incubation period - unknown but estimated at weeks to months. The route of infection is aerosol or direct inoculation with hematogenous spread to the CNS.

• Risk factors - alcoholism, drug abuse, chemotherapy, corticosteroids and organ transplantation

• Presentation:– Symptoms - headache, confusion, fever, lethargy,

nausea and vomiting, seizures, photophobia and neck stiffness. Patients may become frankly psychotic.

– Signs - neck stiffness and focal neurological deficits. Patients may also develop raised intracranial pressure.

Chronic Amoebic keratitis(CAK)

• Chronic amoebic keratitis is a progressive disease of the cornea, which is sight-threatening

• Commonly seen in - immunocompetent patients. However, infection does not confer immunity and reinfection is common.

• Risk factors - poor contact lens hygiene, corneal abrasion or exposure of the eye to contaminated water

• Epidemiology - the incidence of AK is 3 per 100,000 and around 85% of cases occur in people who wear contact lenses

• Presentation - secondary bacterial infection occurs commonly, making it difficult to diagnose.– Symptoms - watering of eyes, eye pain with

photophobia, blurred vision and irritation are common.

• Signs - include conjunctival hyperemia, episcleritis, scleritis and loosening of the corneal epithelium. Rarely, trophozoites can infiltrate the corneal nerve and retina, leading to chorioretinitis

Diagnosis• CSF wet mount (usually

lymphocyte predominance and low glucose)-motile trophozoites

• Culture-Agar plates seeded with E.coli

• Immunofluorescence or polymerase chain reaction (PCR).

• Corneal scrape or biopsy

Treatment

• GAE is treated with pentamidine, usually in combination with one or more of the following: ketoconazole, hydroxystilbamidine, paromomycin, 5-fluorocytosine polymyxin, sulfadiazine, trimethoprim-sulfamethoxazole and azithromycin

• CAK-Therapy should include the cationic antiseptic agents, of which chlorhexidine or polyhexamethylene biguanide (PHMB) is the most effective.

• Ocular lesions –enucleation of ulcer and corneal transplant

Prevention

• killing Acanthamoeba spp. from the contact lens.

• Tap water should not be used to rinse contact lenses

Balamuthia

• It was first identified in 1986 in a specimen from the brain of a baboon that died in the San Diego Wild Animal Park.

• Since then, approximately 200 cases of Balamuthia disease have been reported worldwide.

• Little is known at this time about how a person becomes infected.

Morphology

• Trophozoite- Two forms of pseudopodia either broad lobose or finger like.

- 12-60µm in length - sluggishly motile• Cyst – It is spherical -6-30µm in size

Pathogenicity• Balamuthia amoebas are thought to enter the body

when soil containing Balamuthia comes in contact with skin wounds and cuts, or when dust containing Balamuthia is breathed in or gets in the mouth.

• Once inside the body, the amoebas can then travel to the brain and cause Granulomatous Amebic Encephalitis (GAE).

• GAE is a severe disease of the brain that is fatal in over 95% of cases.

• It can take weeks to months to develop the first symptoms of Balamuthia GAE after initial exposure to the amoebas.

Some early symptoms might include a combination of the following:

• Severe headache• Stiff neck, or neck pain with neck movement • Sensitivity to light • Nausea and vomiting • Unusual fatigue• Fever • Difficulty walking or talking• Sudden one-sided weakness• Behavioral changes• Seizures• Unusual skin lesions that persist over months

Diagnosis• Microscopical examination

of CSF –Trophozoites• Tissue culture• The indirect

Immunofluorescence assay (IFA) is a test used to detect antibodies attached to Balamuthia amoebas in body tissues.

• PCR

Treatment• A combination of

flucytosine, pentamidine, fluconazole, sulfadiazine and either azithromycin or clarithromycin.

• Surgical excision of the lesion may reduced the parasite load.

Sappinia pedata

• Gelman et al. (2001) reported the first and only case of Sappinia amoebic encephalitis in a 38-year-old previously healthy, immunocompetent male .

• It had been isolated from soil, fresh water, forest litter, mammalian faeces and the rectum of lizard.

• It has been described from Europe, North America, Egypt, the Middle East, the West Indies, and Japan

Morphology

• Both trophozoite and cyst stages are binucleate.

• The trophozoite measures 40–80 mm, is ovoid or oblong, and appears to be flattened with occasional wrinkles on the surface.

• The mature cyst is round and measures 15–30mm

Sappinia amoebic encephalitis

• Description - meningoencephalitis associated with cerebral tumor-like lesion, described in one case only.

• Incubation period, mode of spread and risk factors - all remain unknown. It is likely to be reach the CNS either through the nasal mucosa or the bloodstream.

• Epidemiology - only one case described in the literature.

• Presentation - sinus infection was followed by headache, vomiting and photophobia.

Diagnosis

• CT brain scan in the single reported case revealed a tumor-like mass.

• PCR is likely to be a very important tool in diagnosing this particular infection.

Treatment

• In the reported case, the cerebral lesion was surgically removed and azithromycin, pentamidine, itraconazole and flucytosine were also administered.