Pp. 124-149 (26)
Acanthamoeba Species is the most common free living amoeba present in
environment. It is isolated from soil, water, contact lens solutions, transplant units and
various other hospital environment. There are many species of Acanthamoeba such as
A.astronyxis, A.castellani, A.culbertsoni, A.hatchetti, A.keratitis etc. which are known
to cause opportunistic infection in both immunocompetent as well as
immunocompromised host. Transmission mainly occurs through direct contact. There
are two described morphological forms; a trophozoite form and a cyst form. The
trophozoites have characteristics pointed thorn like acanthapodias, containing one
nucleus with central dense large nucleolus. The cytoplasm measures between 15-50μm,
granular and contains various organelles. Clinically patients usually presents with
granulomatous amoebic encephalitis (GAE) which is characterized by focal
neurological deficit, headache, visual disturbances, seizures and behavioral
abnormalities which develops over months to years. Laboratory diagnosis of
Acanthamoeba spp. is done by examining CSF which generally shows predominant
lymphocytes, elevated proteins and low glucose levels. Histopathological samples of
the brain generally reveals cerebral edema, multiple necrotic and hemorrhage lesions.
Acanthamoeba can easily be cultivated on non-nutrient agar with overlay of
Escherichia coli or Entrobacter spp. Amoeba feeds on bacteria’s and confluent growth
is seen in 4-5 days of culturing. The combination therapy is advisable in proven cases
of Acanthamoeba infection. Combining Amphotericin B plus Trimethoprim-
Sulphamethoxazole plus rifampicin has successfully used in few cases.
Naegleria Fowleri: The organism was first reported in Australia in 1965. It is an
environmental ameboflagellate parasite found in variety of water bodies such as ponds,
swimming pools; aquarium etc. prefers temperature of 30-45ºC. There are three stages
seen in Naegleria life cycle: the infective trophozoites, transient flagellated and the
resistant cystic stage. The portal of entity of trophozoites is via olfactory
neuroepithelial cell lining covering the cribriform plate to reach olfactory bulb.
Demyelination and myelinoclasis are observed in gray matter due to vascular blockage.
These pathological changes are attributed to release of phospholipolytic enzymes
which causes breaks in the lipid membrane of neuronal cells. Clinically, patients of
primary amoebic meningoencephalitis (PAM) usually presented with high grade fever,headache, photophobia and features of raised intracranial pressure. Laboratory
diagnosis is done using peripheral smear, CSF examination, culture, histopathological
examination and imaging modalities. Hematological findings are leukocytosis with
predominant neutrophils. The CSF shows low glucose and high protein levels.
Centrifugation of fresh CSF sample up to 500 RPM may reveal motile trophozoites.
Morphologically, the size of trophozoites ranges between 12-25μm, with a single
nucleus and centrally placed nucleolus in the absence of peripheral chromatin. Liquid
culture media such as such as Nelson’s medium containing ox liver digest and glucose
are used with serum for growing amoebae. Mammalian cell lines can be employed to
demonstrate cytopathic effect. Multiplex PCR detects free living amoeba within 6
hours but routine use in diagnostic laboratory is limited due to rarity of finding these
organisms and having high cost of PCR. Brain imaging is easy to perform but restricted
by nonspecific findings such as cerebral edema. Specifically, infraction involving
frontal, orbital and cerebellum area can be observed in few cases of PAM. There is no
optimal treatment regime for Naegleria fowleri. Literature suggests combination
therapy works best with amphotericin, rifampicin and azithromycin.
Balamuthia Mandrillaris: Over 200 cases were reported from South America and
United States. The true prevalence of disease is unknown in south East Asia. Organism
is commonly isolated from soil contact with activities related to soil such as gardening,
agriculture pose risk of acquiring the organism. It was first isolated in 1986 from
baboon brain that died of meningoencephalitis. The portal of entry of the organism is
via cutaneous lesions, nasal mucosa and then subsequent spread to brain. CNS lesions
mimic acanthamoeba encephalitis and have chronic slowly progressive course over
many years. The life cycle of Balamuthia involves two stages: trophozoites and the
cyst. The morphologically variable trophozoites are 12- 60 microns in size containing
single nucleus with large centrally placed nucleolus. Cysts are spherical in shape
measuring 12-30μm and contain a single nucleus with double wall having outer
ectocyst, middle fibrillar layer and inner amorphous endocyst.The trophozoites, cysts
and inflammatory cells are observed in perivascular regions of the infected tissue. In
CSF, elevated protiens, reduced glucose are common findings. Balamuthia spp. can be
grown in tissue cultures such as Monkey Kidney cell lines, Human Lung fibroblast and
Human Brain Microvascular Endothelial cell lines. ELISA test is very specific to detect
high antibodies titers. The antibodies do not cross react with other free living amoebae.
PCR is also highly specific and sensitive test in which primers are developed against
mitochondrial rRNA genes. Recently, real time PCR are developed targeting RNAase P
gene of B.mandrillaris.
Sappinia Species: Two species of Sappinia are well-known cause of CNS infections in
humans, Sappinia diplodea and Sappinia pedata. S.diploidea was first isolated from
lizard faeces. As this parasite is found in animal faeces, persons handing livestock are
at higher risk. Only one known case of Sappinia encephalitis infection reported in
literature. The diagnosis was confirmed on histopathological sample, which showed
necrotizing haemorrhagic inflammation of infected tissue, containing trophozoites. The
trophozoite of Sappinia is characterized by two opposing nucleus with central
flattening. Diagnosis can also be done by amplifying rDNA of both Sappinia diploidea
and Sappinia pedata using SSU primers. The real time PCR can also be used based on
18rRNA gene sequences. Sappinia spp. is cultivated on non- nutrient agar with overlay of Enterobacter or Escherichia coli. Vahlkamphia spp. and Paravahlkamfia francinae
are other emerging free living amoebas that were first isolated from CSF of young
patient who presented with typical symptoms of primary amoebic meningoencephalitis.
Acanthamoeba spp., Balamuthia mandrillaris, Negaleria fowleri,
Paravahlkamfia francinae, Sappinia species, Vahlkamphia spp.
Department of Microbiology, Kasturba Medical College, Manipal University, Karnataka, India.