2_Pathogenic free-living amoeba - 2 - Pharmacy Regular.pdf

Pathogenic parasites

1. AMPHIZOIC AMOEBAE
•‘Amphizoic’ because of their ability to live in
two worlds;
–as free-living organisms or as opportunists
•The term amphizoic has been used for
organisms such as;
- Naegleria fowleri,
- Acanthamoeba species,
- Balamuthia mandrillaris
•They are able to infect humans and animals

2. AMPHIZOIC AMOEBAE
•Ubiquitous in nature, found commonly in soil
and water (swimming pools, tap water, heating
and air conditioning units)

•Feed on bacteria

•Multiply both in the body of a host (endozoic)
and in free-living (exozoic) conditions.

3. AMPHIZOIC AMOEBAE
•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

4. Naegleria fowleri
(is a free-living brain eating amoeba)

Primary Amoebic Meningoencephalitis (PAM)
HABITAT:
Free living in warm fresh water (thermo
tolerant amoeba) or moist soil
Invade meninges, encephalic

5. MORHOLOGY
1.Trophozoite – two forms
a. amoeboid stage.
•Single elongate, actively motile by means of eruptive,
blunt pseudopodia called lobopodia, broad anteriorly,
posteriorly tapered or knoblike pseudopodia with
attached debris
•Measures 19 – 20 μm in length, clear nucleus with large
endosome
•Endoplasm vacuolar and granular, with contractile
vacuoles
•Reproduction is by simple binary fission

6. MORHOLOGY
Amoeboid form
•Distinctive phagocytic structures called
amoebostomes
•Used for engulfment and vary in number
depending on the strain

7. b. Flagellate form : Transient flagellate stage
non dividing.
• rapidly motile
• Appear a few hours after amoeboid
form are transferred to water @ 27-37.
Flagellate forms can persist for 2 days –
pear shaped with 2 flagella at the broad
end.
Non dividing, readily shed flagella and
become amoeboid
Not found in CSF or brain

8. 2. Cystic stages- resistant non-motile.
a. Round, uninucleated, thick smooth double
layered cyst wall about 1μ thick.
b. nucleus not evident, few granules appear
empty
c. 7 - 10 μ
d. Amoeba encyst when conditions are
favourable. Not found in CSF or brain
In tissue sections, amoebae poorly
differentiated, has same staining properties as
tissue cells. Clear zone of shrinkage surrounds
amoebae in section – “Halo”

9. Naegleria fowleri

10. Naegleria fowleri. A, Trophozoites from culture
B, Cysts in wet preparation from culture. Unstained. A case of primary
amebic meningoencephalitis in a Nigerian farmer. Am. J. Trop. Med.
Hyg., 29:21-25.)

11. Life cycle of Naegleria fowleri.

12. Life cycle
•Three-life cycle stages
–The actively amoeboid trophozoite and
multiply by binary fission

–The non-feeding flagellate stage that is
produced when the food supply runs low and
acts as a dispersal stage

–A cyst stage that is formed in response to
adverse environmental conditions

13. Naegleria fowleri. Ameba transforming to flagellate
form, A, and flagellate form, B

14. -
Scaning electron micrographs of stages in the life cycle of Naegleria
fowleri: A, Ameba with a single amoebostome (arrowhead); 8,
flagelIate with two flagella emerging from beneath the anterior
rostrum; C, cyst with a single pore, or ostiole, through which the ameba
Escapes.

15. Trophozoites in brain tissue from two fatal cases of
primary amebic meningoencephalitis. With hematoxylin
and eosin stain, the organisms are readily recognizable by
the presence of large karyosomes. Left, multiple vacuolated
organisms are present; the nuclear structure is not in sharp
focus in all. The ameba in the center of the field has a
prominent karyosome and in contrast to the organisms in
its cytoplasm is not vacuolated.

16. TRANSMISSION
Humans usually become infected when they
swim in infected water.
a)Infective stage is the amoeboid trophozoite
form of the parasite taken in through nose

17. Naegleria fowleri
•Trophozoites encyst under unfavourable
condition and excyst under favourable 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)

18. Pathogenesis of Naegleria fowleri

1. Primary Amoebic Meningoencephalitis

Sinuses - filled with amoebae and
exudates
Brain - soft, amoebae in grey matter
Meninges - hyperemic, purulent

19. Olfactory bulb- congested, ulcerated,
hemorrhagic, necrotic
Spinal Cord - Necrotic
Grey matter - Near Subarachnoid space
filled with amoebae
2. Secondary Infection:- Severe cerebral infection,
extension to lungs, spleen and heart.

20. Primary Amoebic
meningoencephalitis
•N. fowleri causes an acute, fulminating
haemorrhagic meningoencephalitis, principally
in healthy children and young adults with history
of recent exposure to warm fresh water

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

21. Primary Amoebic
meningoencephalitis
•The disease progresses rapidly

•Without prompt diagnosis and intervention,
death usually occurs within a week or less

22. Clinical features
•Non specific and that is,
–fever,
–neck stiffness
–followed by headache,
–photophobia,
–confusion,
–Seizures and
– coma

23. Diagnosis of PAM
1.History of patient swimming in stagnant
or thermal water –3 to 6 days prior onset
of symptoms
2.Amoebae from CSF. Amoebae easily
distinguished from inflammatory cells in
Wright or Giemsa – stained preparations
to identify the trophozoite
3.Indirect immunofluorescence and
immunoperoxidase methods distinguishes
amoebae

24. Diagnosis of PAM
4.Culture CSF, trophic or cystic stages grow
on media and perform Temperature and
salinity test –
1. non pathogenic - 42 and 0.85% salinity
2. pathogenic - 45-46 and 0.4% salinity

N. Fowleri trophozoites are found in CSF and
tissue

25. Diagnosis of PAM

•Diagnosis with X- ray, IHAT in CSF
•Occasionally, a C.T scan may be ordered to
rule out cerebral haematoma
•PCR
•A wet- mount of the CSF for the presence of
actively moving trophozoites

27. Treatment of PAM
1.Not satisfactory as few patients have survived
PAM.
2.Amphotericin B (1 mg/kg/day I.V for several
days, intrathecally, or intraventricularly most
effective)
3.Sulphadiazine + Amphotericin B
4.Ketoconazole (800 mg daily orally for 1
month)
5.Azithromycin, a macrolide antimicrobial, has
been shown to be effective against Naegleria
both in vitro and in vivo

28. Prognosis


1.Poor , fulminant, usually fatal within 7 days of
onset.
2.Survival depends on early detection and
treatment

29. Epidemiology of Naegleria fowleri

USA, Europe, Australia, Ireland, South America
Freshwater lakes, ponds
1.Indoor pools filled with river water, flittered,
heated, Cl
2, 10ppm-predisposing factor
2.Heated indoor pools
3.Dirt lined ponds
4.Water snuffing rituals
5.Pipes exposed to high temperatures
6.Thermal pollution – 45 degrees centigrades
7.Natural hot springs

30. Prevention
1.Prevent contact with stagnant or thermal
water.
2.Hyper chlorination not preventive, could be
predisposing
3.Standard swimming pool no infection
reported in USA
4. Salination when in doubt – 0.7%.

31. Acanthamoeba
=formerly hartmanella
Etiology = Granulomatous amoebic encephilitis
Uveitis, and ulceration of the eye (keratitis)
A. culbertsoni, A. Polyphaga, A. astronyxis
A. castellani, A. hatchetti, A. rhysodes, A. divionensis, A.
lugdunensis and A. lenticulata are implicated in human
diseases.
The important species is A. culbertsoni
Habitat:
Free living
Eye, meninges, skin

32. Acanthamoeba culbertsoni
•Free-living trophozoites and cysts occur in both
the soil and freshwater

•Trophozoites occur only as amoeboid forms

33. Acanthamoeba culbertsoni
•Free-living amoeba
•Lives in water, have been found in soil, sea-
water, sewage, swimming pools, contact
lens, equipment, dental treatment units and
air conditioning systems
•Both forms can be the source of infection
i.e. trophozoite & cyst

34. Morphology of Acanthamoeba
1.Active trophozoite
a. Amoebae irregular in appearance with
several pseudpodia-spine like called
acanthopodia
Amoebae larger than Naegleria
fowleri
b. No flagellate form
c. 30 microns
d. Single nucleus with large endosome

35. Phase-contrast micrograph of leptomyxid trophozoites and
cysts in culture, originally isolated from human brain. Note
extensive branching of trophozoites and wavy appearance
of cysts.

36. Acanthamoeba culbertsoni. A, Trophozoite exhibiting
numerous acanthopodia, a nucleus and a contractile vacuole,
and attached to a cyst with somewhat atypical,endocyst wall.
B, Cyst with typical polyhedral endocyst wall and spherical
ectocyst.

38. 2.Cyst, 20 microns
a. Double walled: quite resistant in the
environment. Cyst wall ectocyst smooth
or slightly wrinkled, endocyst roughly
polyhedral

39. Acanthamoeba. Gross appearance of sectioned from a man who died 3
weeks after onset of meningoenitis. Amebae were found in areas of
necrosis in the midd adjacent structures (above, arrows) and in the
temporal left parietal cortex, and cerebellum (below, arrow). In
Other sections of the brain, cysts as well as trophozoites were present
Am. J. Trop. Med. Hyg. 27:29-38

40. Acanthamoeba. Two trophozoites (arrows) near a small
blood vessel. Am. J. Trop. Med. Hyg., 27:29-38.)

41. Acanthamoeba species, Trophozoites and Cysts. Figure 3. In this hematoxylin and eosin-stained section of
human brain, it is
possible to recognize large numbers of trophozoites (arrows) encircling an arteryeven at this moderate
magnification. Figure 4. At oil immersion magnification four trophozoites are seen in this hema-
toxylin and eosin-stained section; two of the organisms show the characteristic prominent karyosomes.
Figure 5. Trophozoites and a cyst are seen in the same field in this hematoxylin and eosin-stained section of
brain. Both the outer wall and the ~e inn~wall of the cyst are clearly visible. One feature useful in
differe~tiating Naegleria and&anthamoeba infections of the brain is that cysts only occur in tissue with
Acanthamoeba infection. Figure 6. Three cysts of Acanthamoeba spp are seen in this hematoxylin and eosin-
stained section of human brain; they are present in the wall of a vein. The wrinkled appearance of the cyst
wall is clearly evident.

42. Life cycle
•There are two life stages
–The active feeding and dividing trophozoite
stage
–Cyst stage
•Man acquires infection by inhalation of
aerosol or dust and sometimes through broken
skin
•Invade the nasal mucosa and CNS through the
blood stream

43. Life cycle of Acanthamoeba

44. Pathology and Symptomatology
1.All the species are pathogenic
2.Port of entry broken skin or eye, lungs via
breathing when swimming in infected water,
possibly genital or urinary tract
3.Gradual onset of symptoms. Prolonged,
chronic, months/weeks.
4.In chronically ill and immunosuppressed = sore
throat, fever, meningeal irritation, headache –
stiff neck, 3 weeks onset to death.

45. 5. Brain absess, purulent leptomeningitis.
Brain oedema, Necrosis of the thallamus
and cerebellum pons, and frontoparietal
lobes. No olfactory nerves and lobes
involved.
6.Both trophozoites and cysts in lesions:
skin, kidneys, liver, spleen, uterus.
Prostate, pancreas, lungs.
- superficial skin ulcer

46. 7.Eye lesions: muddy water wadding + sore
throat – conjuctivitis, iritis, fever. 7 year
old Texas died.

Brain sections – A castellani
- corneal ulcers
- enucleation (removal of an
organ or tumour in its entirety)
- amoebic keratitis
- recurrent uveitis

47. keratitis : corneal inflammation and corneal perforation often
followed by blindness.

48. Zambian who died of meningoencephalititis
= granulomatous, leptomatoid
Started as lesion at umblicus

-Chronic Acanthamoeba granuloma from
head – Malawi

49. Acanthamoeba spp
•Contact lens wearers can get keratitis (infection
of the cornea ) by using tap water for lens
disinfection or by swimming when wearing
lenses

•When Acanthamoeba spp. enters the eye, it can
cause severe keratitis in otherwise healthy
individuals, particularly contact lens users

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

51. Acanthamoeba spp
•Diseases caused
•1. Granulomatous amoebic encephalitis
(GAE) affect people with compromised
immune systems
•2. Disseminated granulomatous amoebic
disease (e.g. skin, sinus and pulmonary
infections)
•3. Amoebic keratitis (a sight-threatening
disease. Most cases occur in people who
wear contact lenses)

52. Acanthamoeba
•Cysts are common and are very resistant to
chlorine
•Opportunists can cause infections in
humans
•Infections with these amoebae are more
common in immunocompromised patients

53. Diagnosis
•CSF wet mount (usually lymphocyte
predominance and low glucose) – motile
trophozoites
•Culture –agar plates seeded with E. coli
•Immunofluorescence
•PCR
•Corneal scrape or biopsy

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

55. Treatment
•Ocular lesions – enucleation of ulcer and
corneal transplant

56. Ballamuthia mandrillaris:
1.Causes necrotizing, granulomatous
amoebic encephalitis in humans and
animals
2.New world (Peru), Australia, Europe and
possibly Africa )
3.A case report from Zambia.

57. Ballamuthia mandrillaris:
•It was first identified in 1986 in a specimen
from the brain of a baboon that died in San
Diego Wild Animal Park
•Since then, approximately 200 cases of
Balamuthia diseases have been reported
worldwide
•Little is known at this time about how a person
becomes infected

58. Morphology
•Trophozoite
–Irregular or branching in shape
–Length ranges from 12 – 60µm
–Sluggishly motile
–In tissue culture, broad pseudopodia are
usually seen

59. Morphology
•Cyst
–Are spherical 6 – 30 µm with two walls
–Inner wall is thin and spherical
–Outer wall is thick wrinkled
–Both trophozoites and cysts of B. mandrillaris are
found in tissue, but which of these is infective is not
known

60. Pathogenicity
•Balamuthia amoebae 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 amoebae can
travel to the brain and cause GAE

61. Symptoms
•Include;
•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

62. Section of brain with granulomatous amoebic
Encephalitis. This section is from a Zambian patient who died
from AIDS. The parasites, which were first believed to be an
Acanthamoeba species, failed to react with a specific anti-
serum and are probably attributable to a B. mandrilaris. In
this section, the nuclear structure of the invasive tropho-
zoites is clearly seen. The amoebae of both B. mandril/oris and
Acanthamoeba species are also quite often found in skin
abscesses in Patients with AIDS.

Balamuthia mandrillaris infection in a Peruvian
Girl.This girl was believed to have become infected from
swimming in ponds in northern Peru. The organism causes a
necrotising, granulomatous encephalitis in humans and
animals in the NewWorld, as well as in Europe,Australia
and possibly Africa (see 889). Parasites were recovered
from the biopsy site seen on the right cheek. A bilateral,
granulomaous lesion was present on the face and computed
tomo graphy revealed extensive, intracranial masses, which
werepresumed also to be caused by this pathogen.

Trophozoite of Balamuthia mandrillaris
This leptomyxid amoeba was first isolated from a baboon
and has since been found in several cases of granulomatous
amoebic encephalitis in humans. The patients are always
immunodeficient in some way (e.g. through extremes of age
or with HIV infection). The living trophozoites in culture are
extremely irregular in shape and readily distinguished from
those of Naegleria or Acanthamoeba. (SEM)

63. Diagnosis
•Microscopic examination of CSF for trophozoites
•Tissue culture
•The indirect immunofluorescence assay (IFA) is
a test used to detect antibodies attached to
Balamuthia amoebae in body tissues
•PCR

64. Treatment
•A combination of flucytosine, pentamidine,
fluconazole, sulfadiazine and either
azithromycin or clarithromycin
•Surgical excision of the lesion may reduce
the parasite load

65. Thank you