Free living amoebae

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PRESENTED BY-DR ABHISHEK KUMAR JAIN

PG 2ND YEARMICROBIOLOGY DEPARTMENT

G. R. M. C. GWALIOR

Free Living Amoebae

Wednesday, May 3, 2023

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Over view

Introduction.Epidemiology.Classification.Structure and life-cycles.Clinical Manifestations and Pathogenesis.Host immunology.Diagnosis.Treatment.Prevention and control.

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Introduction

Free-living amoebae (FLA) are small, freely living, widely distributed in soil and water.

FLA of the genera Naegleria, Acanthamoeba, Balamuthia and Sappinia can cause disease in humans and other animals.

Normally, they live as Phagotrophs- in aquatic habitats where they feed on bacteria. Opportunists- in humans, they may produce serious infection of the central

nervous system (CNS) and the eye.They are termed as ‘amphizoic’ has ability to live in two worlds, as free-

living organisms and as endoparasites.

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Epidemiology FLAs are aerobic, eukaryotic protists that comprise several genera.

Worldwide Geographic distribution.

Found in fresh water, mud and moist soil and normally feeding on bacteria.

Hundreds of patients with Central nervous system (CNS) invasion by Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris and thousands of Acanthamoeba keratitis has been reported in worldwide.

In India, 2 cases of Primary Amoebic Meningoencephalitis reported by Pan and Ghose in 1971.

In India, >20 cases were reported so far from Mangalore, Kolkata and Rajasthan.

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Classification Kingdom- Protista. Subkingdom- Protozoa. Phylum- Sarcomastigophora. Subphylum- Sarcodina. (has 2 classes)

Class 1- Lobosea (contained two orders) Order- Amoebida

Family- Acanthamoebidae, *Genus- Acanthamoeba.• Species- A. astronyxis, A.

castellanii, A. culbertsoni and A. polyphaga.

Order- Schizopyrenida Family- Valkampfidae,

*Genus- Naegleria.• Species- N. fowleri (in human),

N. australiensis & N. italica (in mice)

Class 2- Acarpomyxea Order- Leptomyxida

Family- Leptomyxidae, *Genus- Balamuthia.• Species- B. mandrillaris.

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Structure and life-cycles

The nuclei of the FLAs are characterized by a large central nucleolus or karyosome, and a nuclear membrane without chromatin granules.

Naegleria -has three stages Trophozoite- an amoeboid form, shows brisk progressive movements at 21oC by means of

rounded pseudopodia (lobopodia). 9- 15µm in diameter, Slud-shaped with one broad and one pointed extremity and known as LIMAX amoebae.

Cyst- dormant form, thick smooth double layered cyst wall. Flagellate form.

Reproduction in is by simple binary fission of the trophozoite. Nuclear division is promitotic- During this process, the nuclear membrane remains intact.

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Naegleria fowleri

7-15µm

“The brain-eating amoeba” first discribed by M. Fowler.

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Acanthamoeba and Balamuthia mandillaris (Leptomyxid FLA) Has two stages and both can be source of infection to man. Trophozoite-

20-50µm in size Has a rough exterior with several spine-like projections (acanthopoda)

Cyst – Has a winkled outer surface with smooth inner wall with large central,

dense nucleolus surrounded by halo.

Acanthamoeba Balamuthia mandillaris (Leptomyxid FLA)

Trophozoite •15-25µm in size, •Spine or thorn like pseudopodia (acanthopodia)•Nucleus- single with central karyosome and no peripheral chromatin.

•~30µm in size,•Irregular with fingure like pseudopodia.

Cysts •Doubled walled (outer wrinkled ectocyst and inner endocyst)•With large central, dense nucleolus surrounded by halo.

6-30µm, surrounded by 3 layered cell wallOuter- wrinkled ectocyst,Middle-mesocyst,Inner-endocyst.

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Sappinia diploidea Newly recognized pathogenic

FLA found insoil and water. Trophozoite- oval, 40-70µm in

size, binucleated. Cyst- round, 15-30µm in size,

and binucleated. Can be cultivated on non-

nutrient agar plate coated with bacteria.

Till now, only one case of amoebic encephalitis has been reported.

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Clinical Manifestations and Pathogenesis

Four distinct clinical syndromes are caused by the FLAs that infect humans: 1. Primary amoebic meningoencephalitis (PAM)- Naegleria

fowleri; 2. Granulomatous amoebic encephalitis (GAE)- Acathamoeba;3. Amoebic keratitis (AK)- Acathamoeba and4. Disseminated granulomatous amoebic disease- Acathamoeba

and Balamuthia. (e.g.- skin, pulmonary, and sinus infection).

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1. Naegleria fowleri -Primary Amoebic Meningoencephalitis

Geographical Distribution: some parts of the world. Usually occurs in otherwise healthy children and young adults Mode of infection:- Swimming and sniffing (inhalation) in contaminated

water. Onset of symptoms -2 to 5 days after exposure, (Apparent IP-upto 2 week).

Diffuse Meningoencephalitis

14Very early involvement of the olfactory nerves changes in taste

or smell an abrupt onset of fever, anorexia, nausea, and vomiting.

Headache and meningismus are noted in 86% to 100% of patients,Mental status changes in 66%.Patients rapidly progress to coma and death within 1 week after

the onset of illness, usually without developing focal neurologic signs.

Only one AIDS patient with Naegleria CNS infection has been reported.

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Pathogenesis and Clinical Picture

Amoeboid trophozoite

Nasal mucosaCribriform plate

Olfactory nerve

Brain, meninges

•Diffuse meningoencephalitis with haemorrhage and necrosis of brain tissue

•Fever, headache, nausea, vomiting, stiffness of neck, convulsions.

•Disturbance in the sense of smell and taste

•Coma and death within 3-6 days from infection

•Thus, Naegleria causes acute fulminant rapidly fatal disease

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brain sectionin vitro culture

18A. Granulomatous Amoebic Encephalitis B. Acanthamoeba Keratitis

Mode of infection•Nose to Lower

respiratory tract to Blood to Brain•Ulcerated skin and mucosa to Blood to Brain

•Through corneal trauma•Exposure to contaminated water•Wearing contaminated contact lenses

Acanthamoeba spp.

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Pathogenesis and Clinical Picture of GAE

- Headache, nausea, vomiting, convulsions, stiffness of the neck and altered mental state.

- Sub-acute or chronic course lasting for weeks to months or years.

- In AIDS patients, the disease may be fulminating resembling infection with Naegleria

- A. culbertsoni and A. castellani are frequently identified species in CSF.

•Acanthamoeba causes single or multiple focal granulomatous space-occupying lesions in the brain.

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Pathogenesis and Clinical Picture Amoebic Keratitis

Mechanism of adhesion- Mannose binding protein on Acanthamoeba adheres to glycoprotein receptors on corneal epithetium.

Characterised by- corneal infiltration and ulcerations, iritis, scleritis, hypopyon, severe pain, and loss of vision.

In india, 75-93% of cases associated with contact lens users.

A. polyphaga and A. castellanii frequently identified species in the corneal scrapping.

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Cyst

Trophozoite

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Diffuse meningoencephalitis.Runs rapidly fatal course

(death within 3-6 days)

History of swimming in natural water or swimming pools.

Infection occurs through: The nasal route cribriform

plateolfactory nerve brain.

Focal, granulomatous, space-occupying lesion.

Runs sub-acute or chronic course (lasts for weeks, months or years)

Not strongly associated with swimming.

Infection occurs in: Lower respiratory tract, ulcerated

skin or mucosa blood stream CNS

Naegleria meningoencephalitis Acanthamoeba encephalitis

Children & young adults

Debilitated Chronically ill low immunity

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4. Balamuthia mandrillaris

Balamuthia can cause disease in both immunocompetent (especially in children) and immunocompromised hosts.

Subacute or chronic granulomatous meningoencephalitis is the most common clinical presentation,

Resulting in death 1 week to several months after the onset of neurologic symptoms.

Important signs and symptoms include fever, headache, nausea, vomiting, seizure, and focal neurologic signs.

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Host immunity1. PAM-

Since the course of infection is fulminant and rapid, patient usually die within 3 to 6 days, no specific antibodies are produced.

Role of cell mediated immunity is also inconclusive.

2. GAE and AK- Intact immune system confers protection against GAE. Impaired humoral and CMI make the person more susceptible.

3. Balamuthia- Can cause disease in both immunocompetent (especially in children) and

immunocompromised hosts.

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Diagnosis

1. Primary Amoebic MeningoencephalitisRecent H/O swimming in thermal or stagnant water.H/O contact with fresh water, mud or dust, 2 to 6 days prior to

onset of symtomes of meningeal irritation.Age of patient= usually children and young patients.Final diagnosis is depends on the detection and identification

of trophozoite of Naegleria in the CSF or biopsied brain tissue.

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Microscopy/Direct examination CSF is specimen of choice. CSF analysis-

CSF is sanguinopurulent shows stronge neutrophilic reaction.

Raised CSF pressure. CSF shows pleocytosis

CSF biochemistry Protein – raised Glucose – normal or low.

28Wet mount- shows active

directional movements.Trichrome, Giemsa and Wright

stains are used to stain the organism in CSF smear.

Direct fluorescent antibody staining is most sensitive method.

Serodiagnosis- not useful.

Culture Confirmed by culture on non-

nutrient agar (Page’s saline and 1.5% Agar) plates spread with gram-negative bacteria (eg; E. coli)

Other culture media- Tryptic soy agar with horse blood, Buffer charcoal yeast extract

(BCYE) Incubation- for 48hr

At 37oC (Naegleria) or At 30oC (Acanthamoeba)

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Culture media

Ingredients Quantity Sodium chloride (NaCl) 120 mgMagnesium sulphate (MgSO4.7H2O) 4 mg

Calcium chloride (CaCl2.2H2O) 4 mgDisodium hydrogen phosphate (Na2HPO4)

142 mg

Potassium dihydrogen phosphate (KH2PO4) 136 mg

Distill H2O 1000ml

Suspend all ingradient in 1000 ml distilled water. Heat if necessary to dissolve the medium completely.

Add 1.5% (15gm) of Agar-agar.

Sterilize by autoclaving at 15 lbs pressure at 121°C for 15 minute.

•Page’s saline with 1.5% Agar medium-

30 “Trail sign” left by migrating amoebae, in the lawn culture can

be visualized following incubation at 37oC for 48hr

Molecular diagnosis- DNA probe and PCR for

identification from clinical and environmental material targrting specific 5.8s rRNA gene.

Useful in postmortem diagnosis and for research purposes.

CT-scan of head- not diagnostic Shows loss of subarachnoid space

and shows diffuse gray matter enhancement.

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2. Granulomatous Amoebic Encephalitis

GAE is rarely diagnosed before death. Most cases have been diagnosed post-mortem or shortly before death. Laboratory diagnosis is always parasitic. Microscopy-

By identifying trophozoite form in CSF wet mount and smear (Acathamoeba & Balamuthia) By identifying Trophozoite and cysts in the brain tissue Both Trophozoite and cysts can be demonstrated in the direct saline wet-mount of corneal

scrapings and biopsy. Acridine orange, Giemsa, LPCB and Parker ink-KOH stain are frequently

used to stain both cyst and trophozoite. Both can be demonstrated by Immunofluorescence using fluorescence-

conjugated lectins (concavalin- A) and wheat germ agglutinin.

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Cyst form stained with H & E

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Trophozoite form stained with H & E

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Contact lens and its saline solution, CSF and biopsy specimen. Inoculated on non-nutrient agar plates spread with gram-negative bacteria

(eg; E. coli) and incubated at 30oC for 48hrs.Serodiagnosis- not useful.CT-scan of head-

Shows multiple luscent, non-enhancing lesions in the cortex of the brain, Focal lesions are common and found through out the CNS.

Other test- CSF shows elevated protein, normal or slightly decreased glucose levels.

With prominent lymphocytic reaction.

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Species identification may be made by using the indirect fluorescent antibody technique (IFAT) and specific antisera against Acanthamoeba spp. or B. Mandrillaris.

The species of Acanthamoeba identified most frequently from cases of GAE have been A. Castellanii and A. culbertsoni.

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Treatment

Although Acanthamoeba keratitis may be treated with antimicrobial agents,

virtually all cases of PAM and GAE have been fatal because there is no effective treatment.

PAM Amphotericin B is drug of choice(i.v. or intrathecally) Miconazole, sulfisoxazole, phenothiazine, rifampin, chloramphenicol,

and tetracycline are also evaluated in treating PAM.

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GAE & AK- No effective therapy is available Acanthamoeba is sensitive to sulphonamides, clotrimazole and

polymyxin B. Drug treatment of AK has more successful than GAE. AK has respond well to topical miconazole antibiotic followed by

Keratoplasty.

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Prevention and control

No vaccine is available.Avoidance of contact with stagnant or thermal water (if N.

fowleri is detected in these sources) may be the only method of prevention.

Even hyperchlorination of swimming pool water is not protective against Naegleria infection.

Preventive measures to GAE is difficult as the amoeba are ubiquitous in air, soil, and water.

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The Amoebic keratitis, caused by contact lens is preventable by means of- Proper cleaning of contact lenses by using commercial rather than

home made saline solutions. Disinfecting contact lenses preferably with a thermal system, and Not wearing lenses while swimming.

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Microbiology, Clinical Characteristics, Diagnosis, and Treatment of Free-Living Amebae

Known to Cause Human Disease

N. fowleriAcanthamoeba spp.

B.mandrillaris(non-keratitis disease) (keratitis)

Disease PAM GAE,Cutaneous lesions,sinus infection

Amoebic keratitis GAE,Cutaneous lesions, sinus infection

Epidemiology Associated with exposure to recreational warm fresh water

Can acquire from soil, water, air

Corneal trauma; poor contact lens hygiene

Can acquire from soil, water, air

At risk Healthy children and young adults, usually male

Immunocompromised individuals

Contact lens wearers(>80% of cases)

Immunocompromised individuals; healthy children and elderly;

Signs & symptoms Headache, neck stiffness,seizures, coma

Headache,neck stiffness, behavioral changes, coma; sinus disease; skin ulcers

Intense pain, photophobia,tearing; dendriformepitheliopathy (early);stromal ring

Headache, neck stiffness,seizures, hydrocephalus; sinus infection; skin nodules

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Clinical course Prodrome of few days;fulminant disease; withouttreatment,death occurs within 1-2 wk

Prodrome of weeks to months; subacute course;acute stage fatal in week

Prodrome of days; subacute to chronic keratitis

Prodrome of weeks to months; subacute course; acute stage fatal in weeks

Laboratory diagnosis CSF wet mount positive formotile amebae; with PMN cells and Pleocytosis;PCR from CSF

Amoebae rarely seen in CSF wet mount;Cysts seen in brainTissue-test by IFA, IIF. PCR for definitive identification

Corneal scraping or biopsy to find trophozoites or cysts confocal microscopy

Amoebae rarely isolated from CSF, but CSF can have highly elevated protein; cysts seen in brain tissue—test by IFA, IIF, and PCR.

Distinct morphologicfeatures

Vesicular nucleus; limacine movement offlagellate stage; cysts with pores at surface

Vesicular nucleus; finger-like pseudopodia projecting from surface; Cyst wall with 2 layers and with pores

Vesicular nucleus with single or multiple nucleoli; ameboid and“spider-like” movements in culture; cyst wall with 3 layers

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Culture Non-nutrient agar with GNB;Tissue culture cellOptimal growth at ≥37° C

Non-nutrient agar with GNB;Tissue culture cells (Monkey kidney cell line, HEp2, Vero and diploid macrophage cell line);Optimal growth at 37° C (CNS isolates) or at 30° C (corneal isolates)

Non-nutrient agar;Tissue culture cells;Optimal growth at 37° C(bacterized medium not useful)

CT/MRI of head Nonspecific Space-occupying or ring-enhancinglesion

Not applicable Space-occupying or ringenhancinglesions

Antimicrobialtherapy

Intrathecal and intravenousamphotericin B, azoles,rifampin, possiblyMiltefosine

Pentamidine, azoles, flucytosine,sulfadiazine, miltefosine,amikacin IV and IT, voriconazole

Polyhexamethylene biguanide (PHMB), chlorhexidine,propamidine, hexamidine,topical and oral Voriconazole

Pentamidine, azithromycin,fluconazole, sulfadiazine,flucytosine, miltefosine

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References

1. Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases, 8th Edition

2. Topley and Wilsons Microbiology and Microbial Infections, Vol. 4 Parasitology, 10th Edition.

3. Textbook of medical parasitology by S. C. Parija 3rd Edition.4. Parasitology by K. D. Chatterjee 13th Edition.5. Essentials of Medical Parasitology by A S Sastry. 1st Edition.6. Primary Amoebic Meningoencephalitis: First Reported Case from Rohtak,

North India; Naveen Gupta et al; The Brazilian Journal of Infectious Diseases 2009;13(3):236-237.

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Free living amoebae