1. The document discusses various aspects of free-living amoebae (FLA) including Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris. 2. It covers their classification, structure, life cycles, modes of infection, clinical manifestations like primary amoebic meningoencephalitis and granulomatous amoebic encephalitis. 3. The summary also discusses their diagnosis through microscopy, culture, molecular and imaging techniques as well as challenges in treatment.

1. PRESENTED BY-
DR ABHISHEK KUMAR JAIN
PG 2ND YEAR
MICROBIOLOGY DEPARTMENT
G. R. M. C. GWALIOR
Free Living Amoebae
1
Tuesday, March 22, 2016

2. Over view
 Introduction.
 Epidemiology.
 Classification.
 Structure and life-cycles.
 Clinical Manifestations and Pathogenesis.
 Host immunology.
 Diagnosis.
 Treatment.
 Prevention and control.
2

3. 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.
3

4. 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.
4

5. 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.
5

6. 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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7. Naegleria fowleri
7
7-15µm
“The brain-eating amoeba” first discribed by M. Fowler.

8.  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.
8
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 wall
Outer- wrinkled ectocyst,
Middle-mesocyst,
Inner-endocyst.

9. 9

10. 10

11. Sappinia diploidea
11
 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.

12. 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 and
4. Disseminated granulomatous amoebic disease- Acathamoeba
and Balamuthia. (e.g.- skin, pulmonary, and sinus infection).
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13. 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
13

14.  Very 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.
14

15. Pathogenesis and Clinical Picture
Amoeboid
trophozoite
Nasal mucosa
Cribriform 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
15

16. 16

17. brain sectionin vitro culture
17

18. A. 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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19. 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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20. Pathogenesis and Clinical Picture Amoebic Keratitis
20
 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.

21. 21

22. Cyst
Trophozoite22

23.  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
plateolfactory 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
23

24. 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.
24

25. Host immunity
1. 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.
25

26. Diagnosis
1. Primary Amoebic Meningoencephalitis
 Recent 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.
26

27. 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.
27

28. 28
 Wet 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)

29. Culture media
29
Ingredients Quantity
Sodium chloride (NaCl) 120 mg
Magnesium sulphate
(MgSO4.7H2O)
4 mg
Calcium chloride (CaCl2.2H2O) 4 mg
Disodium 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.
30

31. 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.
31

32. Cyst form stained with H & E
32

33. Trophozoite form stained with H & E
33

34.  Culture-
 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.
34

35.  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.
35

36. 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.
36

37.  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.
37

38. 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.
38

39.  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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40. Microbiology, Clinical Characteristics, Diagnosis, and Treatment of Free-Living Amebae
Known to Cause Human Disease
40
N. fowleri
Acanthamoeba 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; dendriform
epitheliopathy
(early);
stromal ring
Headache, neck stiffness,
seizures, hydrocephalus;
sinus infection; skin nodules

41. Microbiology, Clinical Characteristics, Diagnosis, and Treatment of Free-Living Amebae
Known to Cause Human Disease
41
N. fowleri
Acanthamoeba spp.
B.mandrillaris
(non-keratitis disease) (keratitis)
Clinical course Prodrome of few days;
fulminant disease; without
treatment,
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 for
motile amebae; with PMN
cells and Pleocytosis;
PCR from CSF
Amoebae rarely seen in
CSF wet mount;
Cysts seen in brain
Tissue-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 morphologic
features
Vesicular nucleus; limacine
movement of
flagellate 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

42. Microbiology, Clinical Characteristics, Diagnosis, and Treatment of Free-Living Amebae
Known to Cause Human Disease
42
N. fowleri
Acanthamoeba spp.
B.mandrillaris
(non-keratitis disease) (keratitis)
Culture Non-nutrient agar with
GNB;
Tissue culture cell
Optimal 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-enhancing
lesion
Not applicable Space-occupying or
ringenhancing
lesions
Antimicrobial
therapy
Intrathecal and intravenous
amphotericin B, azoles,
rifampin, possibly
Miltefosine
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

43. 43

44. 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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45. Thank
You
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