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DPDx - Laboratory Identification of Parasites of Public Health Concern

DPDx - Laboratory Identification of Parasites of Public Health

Concern

Free Living Amebic Infections

[Acanthamoeba spp.] [Balamuthia mandrillaris] [Naegleria fowleri] [Sappinia spp.]

Parasite Biology Image Gallery Laboratory Diagnosis Resources

Casual Agents
Free-living amebae belonging to the genera Acanthamoeba, Balamuthia, Naegleria and Sappinia are rare causes of disease
in humans and animals.

Acanthamoeba spp. and Balamuthia mandrillaris are free-living amebae capable of causing granulomatous amebic
encephalitis (GAE). Acanthamoeba is an important cause of severe keratitis among contact lens wearers and may also
cause cutaneous lesions, particularly in immunocompromised individuals. Because species level identification is not
typically performed on Acanthamoeba cases, the full range of pathogenic species within the genus is not known, but
species known to infect humans include A. byersi, A. castellanii, A. culbertsoni, A. hatchetti, A. healyi, A. astroonyxix,
A. divionensis and A. polyphaga. B. mandrillaris may also occur in association with skin lesions. Naegleria fowleri produces
an acute, and usually lethal, central nervous system (CNS) disease called primary amebic meningoencephalitis (PAM).
Sappinia pedata has been implicated in one human case of amebic encephalitis.

Life Cycle
Acanthamoeba spp.

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Acanthamoeba spp. are ubiquitous in the environment and have been found in a variety of sites, including soil; fresh,
brackish, and sea water; field-grown vegetables; sewage; swimming pools; contact lens supplies; medicinal pools; dental
treatment units; dialysis machines; heating, ventilating, and air conditioning systems; and tap water; mammalian cell
cultures; and vegetables. Acanthamoeba has two stages; cysts and trophozoites in its life cycle and lacks a flagellate
stage. The trophozoites replicate by mitosis (nuclear membrane does not remain intact) . The trophozoites are the
infective forms, although both cysts and trophozoites can enter the body through various means. Entry can occur
through the eye , the nasal passages to the lower respiratory tract , or ulcerated or broken skin . 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. Both Acanthamoeba spp. cysts and trophozoites are found in
tissue.

Balamuthia mandrillaris

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Balamuthia mandrillaris has been isolated from soil and dust, and also from autopsy specimens of infected humans and
animals. B. mandrillaris has two stages, cysts and trophozoites in its life cycle and lacks a flagellate stage. The
trophozoites replicate by mitosis (nuclear membrane does not remain intact) . The trophozoites are the infective forms,
although both cysts and trophozoites gain entry into the body through various means. Entry can occur through the
nasal passages to the lower respiratory tract , or ulcerated or broken skin . When B. mandrillaris 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 . B. mandrillaris cysts and
trophozoites are found in tissue; a few cases have been associated with solid organ transplantation from an infected
donor.

Naegleria fowleri

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Naegleria fowleri has three stages in its life cycle: cysts , trophozoites , and flagellated forms . The trophozoites
replicate by promitosis (nuclear membrane remains intact) . N. fowleri is found in fresh water, soil, thermal discharges
of power plants, geothermal wells, and poorly-chlorinated recreational and tap water. Trophozoites can turn into
temporary non-feeding flagellated forms which usually revert back to the trophozoite stage. Trophozoites infect humans or
animals by penetrating the nasal mucosa, usually during swimming or sinus irrigation , and migrating to the brain
via the olfactory nerves causing primary amebic meningoencephalitis (PAM). Naegleria fowleri trophozoites are found in
cerebrospinal fluid (CSF) and tissue, while flagellated forms are occasionally found in CSF. Cysts are not seen in brain tissue.

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Hosts
There is no major reservoir host as these ameba species are free-living in the environment and are only opportunistically
parasitic. However, such infections have been documented in a variety of animal species.

Geographic Distribution
These amebae occur worldwide in water and soil; below is a summary of the locations where human infections are known
to have occurred:

Acanthamoeba spp.: Acanthamoeba-associated keratitis occurs worldwide predominantly among contact lens users. Cases
of Acanthamoeba-associated GAE have been reported broadly as well.

Balamuthia mandrillaris: Reports of B. mandrillaris infections primarily originate from the Americas, particularly Latin
America, with limited cases reported from Asia, Australia, and Europe. In the United States, many cases have been reported
from southwestern states. Many cases reported from Peru have involved the development of facial skin lesions.

Naegleria fowleri: Cases have been reported on all inhabited continents. Most cases in the United States have been
associated with warm freshwater exposure in southern states. However, there have been cases reported from northern
states in summer months.

The single reported case of infection with Sappinia pedata in a human was identified in Texas.

Clinical Presentation
Acanthamoeba spp.: Disseminated infection typically shows up as inflammation of the lungs or sinuses, and/or skin
infections but has the potential to spread to the brain. GAE has a chronic onset that progressively worsens over a span of
weeks to months. Signs and symptoms are typical of meningoencephalitis and encephalitis, and involve varying degrees of
neurological impairment. There are very few known survivors of GAE.

Acanthamoeba keratitis is most commonly unilateral and individual ocular symptoms are variable. A characteristic circular
stromal infiltrate may become apparent in the iris. Corneal ulceration and scarring can occur if not treated. Although highly
associated with contact lens use, cases occurring in the absence of contact lenses have occurred.

Cutaneous acanthamebiasis presents as a single or disseminated chronic skin lesions, which are most commonly crusted
or ulcerated, they may be indurated or have an eschar. Skin infections caused by Acanthamoeba can appear as reddish
nodules, skin ulcers, or abscesses in the skin. The lesions may be mistaken for fungal or mycobacterial skin infection,
cutaneous amebiasis caused by Entamoeba histolytica or cutaneous leishmaniasis. These may occur with or without
concurrent central nervous system disease.

Balamuthia mandrillaris: Infection with B. mandrillaris involves a similar course to Acanthamoeba-associated GAE. The
disease might appear mild at first but can become more severe over weeks to several months. Often the disease is fatal
with a case fatality rate of more than 89%. Plaque-like skin lesions, most often on the face, particularly in the cheek or nose
area, but sometimes on the torso or limbs may precede the development of neurological symptoms by weeks or months.

Naegleria fowleri: PAM is characterized by severe CNS dysfunction with rapid degeneration caused by hemorrhagic-
necrotizing meningoencephalitis. Unlike GAE, the onset of PAM symptoms occurs quickly following infection (1-9 days;
median 5 days after swimming or other nasal exposure to Naegleria-containing water). Symptoms are similar to bacterial
meningitis, for which it is often mistaken, with deteriorating neurological function and complications. The case fatality rate
is extremely high.

Acanthamoeba spp. cysts.

 

The cysts of Acanthamoeba spp. are typically 10—25 µm in diameter. The cysts have a two-layered wall with pores: a
wrinkled fibrous outer wall (exocyst) and an inner wall (endocyst) that may be hexagonal, spherical, star-shaped or
polygonal. Cysts contain only one nucleus with a large karyosome. Cysts may be found in the brain, eyes, skin, lungs and
other organs.

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Figure A: Cysts of Acanthamoeba Figure B: Cysts of Acanthamoeba Figure C: Cyst of Acanthamoeba

spp. in culture. spp. in culture. sp. from brain tissue, stained with
hematoxylin and eosin (H&E).

Figure D: Cyst of Acanthamoeba Figure E: Cysts of Acanthamoeba

sp. from brain tissue, stained with sp. (green arrows) in tissue,
hematoxylin and eosin (H&E). stained with H&E.

Acanthamoeba spp. trophozoites.

 

Trophozoites of Acanthamoeba spp. are pleomorphic and measure approximately 15—45 µm. They often have multiple
spine-like processes called acanthapodia. Trophozoites contain a large nucleus with a large, centrally-located karyosome
but no peripheral chromatin. There is no flagellated trophozoite stage in Acanthamoeba spp.

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Figure A: Trophozoite of Figure B: Trophozoites of Figure C: Trophozoite of

Acanthamoeba sp. from culture. Acanthamoeba sp. from culture. Acanthamoeba sp. in tissue,
Notice the slender, spine-like Notice the slender, spine-like stained with hematoxylin and
acanthapodia. acanthapodia. eosin (H&E).

Figure D: Trophozoites of
Acanthamoeba sp. in a corneal
scraping, stained with H&E.

Balamuthia mandrillaris cysts.

 

The cysts of Balamuthia mandrillaris are highly similar morphologically to those of Acanthamoeba spp. and are also
typically 10—25 µm (mean 15 µm) in diameter. Generally these cannot be reliably distinguished from Acanthamoeba spp.
without either molecular confirmation or electron microscopy. The cysts have two walls apparent in light microscopy, and
do not have pores: a wrinkled fibrous outer wall (exocyst) and an inner wall (endocyst) that may be variable in shape. A
third layer (mesocyst) is only visible via electron microscopy. Cysts contain only one nucleus with a large karyosome. Cysts
may be found in the brain, skin, lungs and other organs.

Figure A: Cysts of B. mandrillaris. Figure B: Close-up of one of the Figure C: Cyst of B. mandrillaris.
cysts in Figure A.

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Figure D: Cyst of B. mandrillaris in Figure E: Cyst of B. mandrillaris in Figure F: Cyst of B. mandrillaris in

brain tissue, stained with brain tissue, stained with brain tissue, stained with H&E.
hematoxylin and eosin (H&E). hematoxylin and eosin (H&E). Image courtesy of the University
of Kentucky Hospital, Lexington,
Kentucky.

Figure G: Cyst of B. mandrillaris in Figure H: Cysts of B. mandrillaris Figure I: Cyst of B. mandrillaris in

brain tissue, stained with H&E. in brain tissue, stained with H&E. brain tissue, stained with H&E.
Image courtesy of the University Image courtesy of Cook Children’s Image courtesy of Cook Children’s
of Kentucky Hospital, Lexington, Hospital, Fort Worth, Texas. Hospital, Fort Worth, Texas.
Kentucky.

Balamuthia mandrillaris trophozoites.

 

Trophozoites of Balamuthia mandrillaris are pleomorphic and measure approximately 15—60 µm. They often produce long
pseudopodia (broader than those of Acanthamoeba spp.). Trophozoites contain a large nucleus with a large, centrally-
located karyosome but no peripheral chromatin. Binucleate forms are rare. There is no flagellated trophozoite stage in B.
mandrillaris.

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Figure A: Trophozoite of B. Figure B: Trophozoite of B. Figure C: Trophozoite of B.

mandrillaris in culture. mandrillaris in culture. mandrillaris in culture.

Figure D: Trophozoite of B.
mandrillaris in culture.

Figure E: Several trophozoites of Figure F: A single trophozoite

B. mandrillaris in brain tissue, (green arrow) of B. mandrillaris in
stained with hematoxylin and brain tissue, stained with H&E.
eosin (H&E).

Figure G: A single trophozoite

(black arrow) of B. mandrillaris in
brain tissue, stained with H&E.

Naegleria fowleri cysts.

 

Naegleria fowleri does not form cysts in human tissue. Cysts in the environment and culture are spherical, 7-15 µm in
diameter and have a smooth, single-layered wall. Cysts have a single nucleus.

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Figure A: Cyst of N. fowleri in

culture.

Naegleria fowleri trophozoites.

 

There are two forms of trophozoites in Naegleria fowleri: ameboid and ameboflagellate, the latter of which is only rarely
found in humans (within CSF). The ameboid trophozoites measure 10-35 µm but when rounded are usually 10—15 µm in
diameter. In culture, trophozoites may grow to over 40 µm. The cytoplasm is granular and contains many vacuoles. The
single nucleus is large and has a large, dense karyosome and lacks peripheral chromatin.

Figure C: Ameboflagellate
trophozoite of N. fowleri.
Figure A: Trophozoite of N. fowleri Figure B: Trophozoites of N.
in culture. fowleri in culture.

Figure D: Trophozoite of N. fowleri Figure E: Trophozoite of N. fowleri

in CSF, stained with hematoxylin in CSF, stained with trichrome.

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and eosin (H&E). Image courtesy of the Texas State

Health Department.

Sappinia spp. cysts and trophozoites.

 

Sappinia is a genus of free-living amebae rarely isolated from human specimens. The genus is found worldwide and has
been isolated in the feces of many animals, including mammals and reptiles. Cysts and trophozoites both possess two
nuclei.

Figure A: Cyst of Sappinia sp. in Figure B: Cysts of Sappinia sp. in Figure C: Trophozoite of Sappinia
culture, viewed under differential culture, viewed under differential sp. in culture, viewed under DIC
interference contrast (DIC) interference contrast (DIC) microscopy.
microscopy. microscopy.

Figure D: Trophozoite of Sappinia Figure E: Trophozoite of Sappinia Figure F: Four trophozoites

sp. in culture, viewed under DIC sp. viewed under DIC microscopy. (yellow arrows) of S. pedata in
microscopy. brain tissue, stained with
hematoxylin and eosin (H&E). In
three of the amebae, the two
nuclei can easily be seen.

Indirect immunofluorescence (IIF) assay for free-living amebic infections.

 

Indirect Immunofluorescence (IIF) assay for the detection of free-living amebic infections.

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Figure A: IIF of Acanthamoeba sp. Figure B: IIF of Acanthamoeba sp. Figure C: IIF of Balamuthia
viewed under UV microscopy. This viewed under UV microscopy. This mandrillaris in brain tissue,
image was taken at 400x image was taken at 1000x oil viewed under UV microscopy.
magnification. magnification.

Figure D: IIF of Naegleria fowleri in Figure E: IIF of Naegleria fowleri in

brain tissue, viewed under UV brain tissue, viewed under UV
microscopy. This image was taken microscopy. This image was taken
at 200x magnification. at 1000x oil magnification.

Gross pathology images in free-living amebic infections.

 

Gross tissue and pathology specimens from free-living amebic infections.

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Figure A: Gross specimen of brain Figure B: Gross specimen of brain

tissue from a patient who died of
granulomatous amebic
encephalitis (GAE) caused by
Balamuthia mandrillaris. The
autopsy specimen revealed
extensive necrotizing (mixed
inflammatory, occasional giant
cells, vasculitic) granulomatous
encephalitis with a subependymal
necroinflammatory process.
Image courtesy of Cook Children’s
Hospital, Fort Worth, Texas.
tissue from a patient who died of
granulomatous amebic
encephalitis (GAE) caused by
Balamuthia mandrillaris. The
autopsy specimen revealed
extensive necrotizing (mixed
inflammatory, occasional giant
cells, vasculitic) granulomatous
encephalitis with a subependymal
necroinflammatory process.
Image courtesy of Cook Children’s
Hospital, Fort Worth, Texas.

Laboratory Diagnosis
 

Acanthamoeba spp.: Acanthamoeba infection can be diagnosed by detection of trophozoites and cysts on microscopic
examination of stained smears of biopsy specimens (brain tissue, skin, cornea) or of corneal scrapings. Lactophenol blue,
acridine orange, silver, and calcofluor white stains have been used in the diagnosis of acanthamoebiasis on histologic
sections and environmental samples (i.e. pelleted contact lens case contents). In granulomatous amebic encephalitis cases,
trophozoites and cysts are only rarely found in the CSF. Acanthamoeba can be cultured from clinical and environmental
samples in the laboratory on non-nutrient agar with a Page’s saline and Escherichia coli overlay.

Confocal microscopy or cultivation of the causal organism, and its identification by direct immunofluorescent antibody,
may also prove useful. When there are large numbers of cysts and/or trophozoites, as in very severe cases, diagnosis may
be possible through direct microscopy on corneal scrapings or contaminated surfaces (i.e. contact lens cases). An
increasing number of PCR-based techniques (conventional and real-time PCR) have been described for detection and
identification of free-living amebic infections in the clinical samples listed above. Such techniques may be available in
selected reference diagnostic laboratories.

Balamuthia mandrillaris infection is generally diagnosed post-mortem. Outside of molecular detection via PCR and recently,
metagenomic deep sequencing, B. mandrillaris is most reliably detected via immunofluorescence or immunoperoxidase
staining of tissue samples.

For Naegleria fowleri infections, the diagnosis can be made by microscopic examination of cerebrospinal fluid (CSF). It may
be possible to detect motile trophozoites on a wet mount, and a Giemsa-stained smear will show trophozoites with typical
morphology. PCR may be used to detect evidence of N. fowleri in CSF and brain tissue. Amebic culture on non-nutrient agar
plates overlaid with a fine lawn of Escherichia coli enhances the likelihood of detection by microscopic methods. (More on
diagnosis of N. fowleri infections).

Real-Time PCR

A real-time PCR was developed at CDC for identification of Acanthamoeba spp., Naegleria fowleri, and Balamuthia
mandrillaris in clinical samples.* This assay targets 18S small subunit ribosomal RNA gene sequences, and uses distinct
primers and TaqMan probes for the simultaneous identification and differentiation of these three parasites.

Reference:

* Qvarnstrom Y, Visvesvara GS, Sriram R, da Silva AJ. Multiplex real-time PCR assay for simultaneous detection of
Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri. J Clin Microbiol 2006;44(10):3589-3595.

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Laboratory Safety
Free living amebae could pose a potential safety risk to laboratory staff, particularly when large numbers are present in
culture. Personal protective equipment (laboratory gown and gloves with eye protection) should be worn at all times, and
other appropriate BSL-2 precautions should be taken. Individuals with immunosuppressive illnesses or open wounds in
exposed areas of skin may be at greater risk of infection when working with cultures of free living amebae.

Suggested Reading
 

Naegleria fowleri: Siddiqui, R., Ali, I.K.M., Cope, J.R. and Khan, N.A., 2016. Biology and pathogenesis of Naegleria fowleri.
Acta Tropica, 164, pp.375-394.
Further information and FAQ on Naegleria fowleri

Acanthamoeba spp.: Lorenzo-Morales, J., Khan, N.A. and Walochnik, J., 2015. An update on Acanthamoeba keratitis:
diagnosis, pathogenesis and treatment. Parasite, 22.
Further information and FAQ on Acanthamoeba spp.

Balamuthia mandrillaris: Lorenzo-Morales, J., Cabello-Vílchez, A.M., Martín-Navarro, C.M., Martínez-Carretero, E., Piñero, J.E.
and Valladares, B., 2013. Is Balamuthia mandrillaris a public health concern worldwide?. Trends in Parasitology, 29(10),
pp.483-488.
Further information and FAQ on Balamuthia mandrillaris

Sappinia spp.: Walochnik, J., Wylezich, C. and Michel, R., 2010. The genus Sappinia: History, phylogeny and medical
relevance. Experimental Parasitology, 126(1), pp.4-13.

Culture methodologies: Schuster, F.L., 2002. Cultivation of pathogenic and opportunistic free-living amebas. Clinical
Microbiology Reviews, 15(3), pp.342-354.

Page last reviewed: August 23, 2019

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