Acta
Neuropathologica

Acta Neuropathol. (Berl.) 49, 7-12 (1980)

9 Springer-Verlag 1980

Granulomatous Encephalitis, lntracranial Arteritis,
and Mycotic Aneurysm Due to a Free-living Ameba
A. Julio Martinez 1, Cirilo Sotelo-Avila 2, Hilda Alcal~ 3, and Eddy Willaert 4
1 PathologyDepartment (Neuropathology),Presbyterian-UniversityHospital, Universityof Pittburgh, Pittsburgh, PA 15213, USA
2 NorthwesternUniversitySchool of Medicine, Head, ClinicalPathology,The Children's Memorial Hospital, Chicago, IL 60614, USA
3 Director of Pathology (Neuropathologist),Hospital Infantil de Mexico, Mexico 7, D.F.
4 MedicalResearch, 151, Veteran's AdministrationMedical Center, Gainesville,FL 32602, USA

Summary. Primary amebic meningoencephalitis and
granulomatous amebic encephalitis are well recognized
clinicopathological entities caused by free-living amebas. Associated arteritis and "mycotic aneurysms" with
infiltration of intracranial arteries by lymphocytes,
amebic trophozoites and cysts have not been previously
reported.
A 26-month-old girl had a 3-week history of encephalitis, characterized, initially, by vomiting and lowgrade fever. Subsequently, she developed ataxia, generalized weakness, lethargy, and esotropia. The first CSF
showed 490RBC/gl, 705WBC/gl with 90% mononuclears. Her pupils reacted briskly to light. Moderate
nuchal rigidity, nystagmus, fixed downward gaze,
anisocoria, bilateral 6th nerve palsy, left arm monoparesis and left Babinski were present. CAT scan
revealed slight symmetrical dilatation of anterior horns
of lateral ventricles and an area of abnormal enhancement above the 3rd ventricle. She died 14 days after
admission, 5 weeks after onset of symptoms.
The brain showed focal necrotizing encephalopathy, involving thalami, cerebellum, brain stem, and
cervical and upper thoracic spinal cord. Numerous
free-living amebic trophozoites and cysts were present
within a chronic granulomatous encephalitis. There
were thrombosis of basilar, posterior cerebral, and
vertebral arteries with profuse chronic panarteritis,
fibrinoid necrosis, and mycotic aneurysms.

Key words: Free-living amebas - Encephalitis Granuloma - Acanthamoeba - Naegleria - "Mycotic aneurysm"

Offprint requests to." A. Julio Martinez, M.D. (address see above)

Increasing interest in the pathogenicity and the biological behavior of free-living amebas belonging to the
genera Naegleria and Acanthamoeba, have been noted
recently in the medical literature. However, the disease
may be very old and unrecognized cases may have
occurred [8].
The most frequent etiologic agents responsible for
primary amebic meningoencephalitis (PAM) and
granulomatous amebic encephalitis (GAE) are freeliving amebas of the genera Naegleria [4, 5, 9, 20] and
Acanthamoeba [2, 10, 11, 13, 18,27,28] or closely related genus [7]. To date, more than 100 cases of PAM
and nearly 20 cases of GAE have been reported. Some
"extracerebral" infections have also been found due to
Acanthamoeba sp. [3, 12, 16, 25].
PAM usually occurs in healthy, young individuals
with a recent history of swimming in man-made lakes,
heated swimming pools, and warm springs. Apparently
the infecting cysts or trophozoites might be transported
by the air [15]. Free-living amebas have been isolated
from the air, [14] from drinking water [22] from the
nasal passages and throats of clinically normal individuals [30, 33], or as contaminant in bacterial [29]
and tissue cultures [24]. Naegleria fowleri produces an
acute, fulminant, necrotizing hemorrhagic meningoencephalitis affecting the olfactory bulbs, base of the
frontal and temporal lobes, and cerebellum. The portal
of entry is through the olfactory neuroepithelium by
direct penetration of sustentacular cells and invasion of
the amyelinic submucosal nervous plexuses by the
trophozoites. No cysts are found in the lesions.
GAE due to Acanthamoeba sp. is probably an
opportunistic infection [21] of the CNS which has
occurred in chronically ill and immunosuppressed
patients without a history of recent swimming. The
portal of entry into the CNS may be from the lower

0001-6322/80/0049/0007/$1.20

8
respiratory tract or f r o m a skin lesion, reaching the
CNS t h r o u g h the h e m a t o g e n o u s route. The predom i n a n t host reaction is a s u b a c u t e or chronic g r a n u l o m a t o u s encephalitis c o m p o s e d of lymphocytes, p l a s m a
cells, monocytes, a n d m u l t i n u c l e a t e d giant cells, hence
the n a m e g r a n u l o m a t o u s amebic encephalitis. Cysts, as
well as trophozoites, m a y be f o u n d within the CNS
lesions. The most affected areas are the m i d b r a i n , basal
ganglia, a n d posterior fossa structures.
Because p a t h o g e n i c free-living a m e b a s are wide=
spread in nature, a n d m a n is frequently exposed to
them, p u b l i c health implications are obvious.
We report a case of g r a n u l o m a t o u s amebic encephalomyelitis in a 2 6 - m o n t h - o l d girl, which was n o t
caused by Naegleria a n d p r o b a b l y n o t by Acan-

thamoeba.

Report of a Case
A 26-montfl-old girl was admitted to the hospital with abrupt
vomiting and low-grade fever.
Two days before her illness began, she waded in Nachez river,
Texas. Three weeks before admission, her coordination was impaired
and she became less active. Eight days before admission, she was
admitted to a hospital for a day because of vomiting, lethargy,
weakness, and esotropia of the left eye. She was given Donnatal and
discharged. Four days later she was readmitted with persistent signs
and symptoms, and generalized, red skin welts. Two days after
admission the skin lesions disappeared.
The cerebrospinal fluid (CSF) examination showed 490 RBC/gl
and 705 WBC/~tl with 90 % mononuclears. On the second hospital
day the CSF revealed 97 RBCAd, 122 WBC/lalwith 99 ~ mononuclears; protein, 322 mg/dl; and glucose, 20 mg/dl. She failed to
improve after administration of Doxycyclineand was transferred to
another hospital.
Examination at the children's hospital revealed a temperature of
38.4~ pulse ll6/min, respirations, 36 rain, and blood pressure,
9'0 mm/Hg, systolic. Moderate nuchal rigidity was noted. The left
pupil was 1 mm and the right was 2 mm; they reacted briskly to light.
There was downward nystagmus, downward gaze deviation, bilateral
6th nerve palsy and a Babinski sign on the left. She was obtunded.
The hematocrit was 33%; WBC, 8,200/gl with 61% segmented
neutrophils; electrolytes, glucose, and BUN were normal. The CSF
had an opening pressure of 225 mm/HzO; 62 RBC/gl, 280 WBC/pl
with 94% mononuclears; protein 350mg/dl and glucose, 24mg/dl.
Gram stain and india ink preparation were negative. Amebas were
searched for, bm not identified in a wet preparation of CSF. Counter
immunoelectroptloresis on sermn and CSF was negative for
Haemophilus influenzae b; Neisseria meningitidis A, C, and D; and
PneurnococcusOmni A through F. Fluorescent staining of smears of
sediment of urine, CSF and gastric aspirate were negative for acid fast
bacilli. Complement fixation titers for cytomegalovirus, herpes, and
mycoplasma were 1:32, 1:8, and 1:8, respectively. The spot screen
and presumptive tests for infectious mononucleosis were negative.
Blood and CSF cultures were subsequently negative. Chloromycetin,
100 mg/kg in four daily doses, Isoniazid, 20 mg/kg/day in two daily
doses, and Rifampin, 20 mg/kg/day in two daily doses were
administered.
An EEG 1 day after admission showed diffuse slow wave activity
without focality. Computerized axial tomography (CAT) showed
asymmetricalventricnlar dilatation and, with the addition of contrast
material, a midline area of enhancement was present above the third
ventricle consistent with an inflammatory or vascular lesion.

Acta Neuropathol. (Berl.) 49 (i980)
She continued to deteriorate, responded only to deep painful
stimuli and required assisted ventilation on the third day of hospitalization. Decadron, 3 mg, four times a day, was administered without
relief. Chloramphenicol was discontinued. The following day, the
serum electrolytes were as follows: sodium, 125 mEq/l; potassium,
3.8 mEq/l; chloride, 88 mEq/1,and CO2, 24 mEqfl. Serum osmolality
was 262 mOsm/1; urine electrolytes and osmolality were compatible
with SIADH, which persisted for 5 days. The CSF, 6 days after
admission revealed an opening and closing pressure of 100, 72 mm
H20, respectively;WBC, 1t0 with 100 % mononuclears, red cells, i ;
glucose, 17 mg % (simultaneous blood glucose 91); and protein, too
high to read. Indirect immunofluorescent antibodies for Herpes
simplex were negative.
Chloramphenicol was reinstated on the 7th hospital day. A
second CAT scan showed an area of low density which did not
enhance in the temporoparietal area. The right lateral ventricle was
effaced. The midline abnormal enhancement above the third ventricle
remained unchanged.
On the 10th hospital day, she remained unresponsive without
brain stem reflexes. Rifampin was discontinued, and Streptomycin,
20 mg/kg/day was administered.
On the 13th hospital day, no evidence of cerebral electrical
activity was detected on the EEG.
She died on the 14th hospital day, 5 weeks after the onset of fever
and vomiting.

Autopsy Findings
The m a j o r pathological findings were limited to the
central n e r v o u s system (CNS) a n d lungs.
The b r a i n weighed 1,310g. The external appearance of the cerebral hemispheres show large areas
o f encephalomalacia. The leptomeninges were transp a r e n t on the convexities a n d thick a n d o p a q u e at the
base. B o t h olfactory nerves were necrotic at their tips.
The d i e n c e p h a l o n a n d walls o f the third ventricle
were necrotic. The t h a l a m u s showed two abscesses, the
largest measured 1.5 cm in diameter. H o r i z o n t a l sections of the b r a i n stem reveals e n c e p h a l o m a l a c i a of the
superior a n d inferior colliculi. The cerebral a q u e d u c t
c o n t a i n e d necrotic debris. The lower p o r t i o n of the
b r a i n stem exhibited h e m o r r h a g i c necrosis o f the e n t i r e ,
floor of the f o u r t h ventricle, the latter being occupied
by necrotic tissue from the cerebellar vermis. The
cerebellum shows diffuse encephalomalacia, more prom i n e n t in the anterior p o r t i o n with p r o t r u s i o n into the
fourth ventricle. H o r i z o n t a l sections o f the spinal cord
showed some foci of m y e l o m a l a c i a at the cervical a n d
u p p e r thoracic levels,

Histopathological Findings
There was a necrotizing " g r a n u l o m a t o u s " encephalitis
involving the t e g m e n t u m , associated with perivascular
cuffing by lymphocytes a n d occasional p l a s m a cell.
There was m i n i m a l chronic leptomeningitis.
The basilar, posterior cerebral, a n d p o r t i o n s of the
vertebral arteries revealed ~
a n e u r y s m s " with
panarteritis (Fig. 1). I n addition, there were segmental

A. J. Martinez et al. : Granulomatous Amebic Encephalitis

9

Fig'. 1. Portion of the wall of the basilar artery. Two multi-nucleated
giant cells are seen in the lumen (arrowheads). Amebic trophozoites
are seen (arrows)between spaces of the media. The interna elastica
lamina is intact in this segment. H.-E. • 490

Fig. 2. t~hrastructmal features of an amebic trophozoite. • 5,400.
lnset: Amebic trophozoites and modest lymphocytic infiltration in
thalamic lesion. H.-E. x 360

organizing thrombosis with multinucleated giant cells
and numerous amebic trophozoites within the arterial
wall, mainly in the adventitia and muscularis. There
were numerous multinucleated giant cells in the
thrombus and in the adventitia (Fig. 1).
Foci ofnecrotizing granulomatous encephalitis, on
the posterior and lateral portions of the medulla
oblongata and in one inferior olivary nucleus were
identified. There was "fibrinoid" necrosis of blood
vessels. Clusters of trophozoites around blood vessels
and within neuropil with virtual absence of inflammatory reaction was also a striking feature. Well
presm'ved amebic trophozoites were seen measuring
1 5 - 20 l~m in diameter. Occasional spherical amebic
cysts were seen with slight wrinkled walls, measuring
10 btm in average diameter. The roots of the 12th
cranial nerve were surrounded by lymphocytes and few
plasma cells.
The cerebellar peduncles and the central white
matter o f the cerebellum, revealed a necrotizing granulomatous encephalitis with multinucleated giant cells,
occasional amebic trophozoites, and a few amebic
cysts. Fibrinoid necrosis of medium-sized arteries was
also seen.

The thalamus showed a large area of necrotizing
"chronic" granulomatous encephalitis with clusters of
amebic trophozoites (Fig. 2, Inset). The choroid plexus
was undergoing necrosis.
Amebic trophozoites were seen in the deeper portions of the Virchow-Robin spaces.
The cervical and upper thoracic spinal cord disclosed focal necrotizing granulomatous myelitis with
mild leptomeningitis. Trophozoites were seen in these
regions. Perivascular cuffing by lymphocytes was prominent. The roots of the cauda equina showed mild
chronic inflammation.

General Histopathological Findings
The lungs showed foci of atelectasis and occasional loci
of chronic pneumonitis, one of them containing an
amebic trophozoite. The rest of the organs showed no
significant pathology.

Special Pathology Studies
Despite p o s t m o r t e m autolysis, trophozoites and cysts
were adequately preserved, permitting ultrastructural

10

evaluation. The technique used was described elsewhere [7].
Ultrastructurally, the trophozoites possessed the
characteristic dense nucleolus surrounded by granular
chromatin and abundant cytoplasm containing short
cytoplasmic processes bound by a thin, well-defined
membrane (Fig. 2). The cytoplasm contained round
mitochondria, free ribosomes, and different-sized vesicles and vacuoles. Some trophozoites revealed abundant, parallel stalks of endoplasmic reticulum, lysosomes, and glycogen-like granules. Golgi apparatus
was not detected. Their nuclei were centrally or eccentrically located, and contained a dense nucleolus.
"Vacuoles" and "dense" bodies were seen within the
nuclear chromatin. The nuclear membrane was double,
but was absent in the dividing trophozoites. In some
instances, the nucleus contained a dividing nucleolus
without nuclear membrane and at other times, two or
three well defined nucleoli were seen. The nuclear
divisions appear to be mesomitotic or at the end of the
telophase. Some trophozoites which may be in a preencystment stage disclosed a condensation of cytoplasm at the periphery and the presence of numerous,
small electron-dense vacuoles, perhaps representing
fragments of cytoplasmic membranes. No interzonal
bodies or nuclear caps were detected.
Cysts were rounded or slightly ovoid, with a
prominent, thick, slightly wrinkled poreless wall often
composed of partially "split" concentrical layers of
electron dense material. In between the splitting there
was finely granular material. Smooth and rough endoplasmic reticula were barely visible. Abundant
electron-dense lysosomal-like bodies were seen in the
cysts.

Microbiologic Studies
The sediment of the CSF collected on the patient's sixth
hospital day was placed into bacterized agar plates at
37~ and 25~ no growth was obtained after 14 days.
Fragments of brain taken at autopsy were placed in
Fletcher's medium and then into bacterized agar plates
at 37~ and 25 ~C. No growth was obtained in either
case.

Indirect Imrnunofluorescent Antibody Analyses ( IFA)
The histologic Sections of the patient's brain were
deparaffinized before application of the diluted antisera. The following antisera, which were produced in
rabbits, were used in this case: (1) Whole cell antisera
[35]; anti-N.fowIeri (ITMAP 359), anti-A.polyphaga
(P 23), anti-A, astronyxis (Ray), anti-A.palestinensis
(Reich), anti-A, royreba (Oak Ridge), anti-A, rhysodes
(CCAP 1534), anti-A, griffini (CCAP 1501/4), anti-

Acta Neuropathol. (Berl.) 49 (1980)
Table 1. Results of indirect immunofluorescent antibody test on
cerebral tissue
Antisera

Dilution

ResulP

Homologous
end-point
titer

0
0
0
0
0
0
0
0
0
0

1/512
1/128
1/64
1/128
1/128
1/128
1/64
1/128
1/128
1/512

0
0
0

1/512
1/512
1/1024

1. Whole cell antisera:

anti-N.fowleri
anti-A.polyphaga
anti-A, astronyxis
anti-A.palestinensis
anti-A, royreba
anti-A, rhysodes
anti-A.griffini
anti-H, vermiformis
anti- V. avara
anti-E, histolytica

1/32
1/16
1/8
1/16
1/16
1/16
1/16
1/32
1/32
1/64
2. Plasma membrane antisera:
anti-A, eulbertsoni
1/32
anti-A, eastellannii
1/64
anti-A, rhysodes
1/64
a 0 = no fluorescence

H. verrniformis (CCAP 1534/7), anti-V, avara (CCAP
1588/A), and anti-E, histolytica (HK 9); (2)Plasma
membrane antisera [31]; anti-A, culbertsoni (A-l), antiA. castellanii (Neff), and anti-A, rhysodes (CCAP
1534/3).
The histologic sections were incubated and the
immune complexes were visualized after incubation of
the sections with fluorescein-conjugated rabbit antiimmunoglobulin (1:50) (Institute Pasteur, France).
The sections were read with a Leitz Ortholux microscope equipped for fluorescence with a ploemopak 2
and a super-pressure mercury lamp HBO 6, 100 W. The
filter system for FITC combined with two interference
filters KP, 490, K 510, and K 445 were used.
The results of the indirect immunofluorescence
analyses are given in the Table 1. None of the antisera
used stained the ameba in the brain sections.

Discussion

The clinical picture and the histopathological reaction
in CNS resembled those of Acanthamoeba, i.e., chronic
granulomatous inflammation with multinucleated
giant cells, lymphocytes, and plasma cells. Such
pathologic findings have not been described in proven
cases of Naegleria. Human and animal experiments are
consistent with these findings [6, 17, 19]. Also, the
presence of cysts has not yet been described in PAM due
to Naegleria; however, the absence of such may be
related to the lack of an identified chronic stage of this
disease.
A very important feature of this case is the arterial
involvement. In some places "organizing" thrombi

A. J. Martinez et al. : Granulomatous Amebic Encephalitis
were seen with a moderately severe panarteritis associated with lymphocytic infiltration and the presence
of foreign body giant cells, There were at least two
places (basilar artery and posterior cerebral artery) in
which "mycotic" aneurysms were present. Molinari
[23] stated that infection of the arterial wall in cases of
"mycotic" aneurysms is mediated through stasis and
sepsis in the vasavasorum. In addition, the inflammation appears to proceed from the adventitial surface
toward the media. Bell and Butler [1] believe that
embolic material, usually from the heart, occludes the
vasavasorum allowing bacterial growth within the wall
of the artery. The inflammatory response weakens the
vessel wall resulting in aneurysmal dilatation and
possible rupture. Some intracranial "mycotic" aneurysms, usually of extravascular origin, may become
thrombosed and may decrease in size or spontaneously
disappear [32]. It appears that free-living amebas, in
particular Acanthamoeba sp., have a predilection for
attacking vascular walls, thus leading to necrotizing
arteritis with thrombosis and aneurysm. The nuclear
division of the amebas in this case appeared to be
mesomitotic; that is, the nuclear membrane disappears
throughout mitosis and polar caps are absent. Mesomitosis division is consistent with Acanthamoeba or
Hartmannetta. The absence of interzonal bodies during
mitosis serves to exclude the Naegleria, as well as
Vahlkarnpfia as the causative organisms. The presence
of the cyst in the patient's CNS also points against
Naegleria since, characteristically, this ameba does not
form cysts in tissues. Furthermore, the cysts present in
the patient's CNS had wrinkled, concentrically laminated walls without identifiable pores. Naegleria cysts
are smooth and contain pores in their double wall.
However, such cysts do not completely exclude
Naegleria, as Page [26] has pointed out that the
morphology of cysts of Naegleria can vary considerably, from smooth to rough to angular and may even
be wrinkled. The large dark karyosome excludes, with
the exception of Iodamoeba, members of the family
Entamoebidae. If the organism identified belonged to
the Entamoebidae family, ultrastructural evidence of
peripherally located chromatin within the nuclei, as
well as an absence of mitochondria, should have been
noted. Such was not the case [26]. The genus Iodamoeba
cannot be completely excluded, but the cysts were .not
typical of that organism.
The results of the immunological studies gave
further evidence that the unknown pathogenic ameba
was neither Naegleria nor Acantharnoeba. Other genera
belonging to the family of Vahlkampfiidae, such as
tetramitus and paratetramitus, might be considered
also, as they have the same mitotic pattern. Organisms
in the tissues of the patient reported herein did not
fluoresce when exposed to anti-N.fowleri fluorescent

11
tagged antisera. Tissues containing N.Jowleri obtained
from autopsy specimens of culturally proven Naegleria
PAM cases stained well with anti-N.fowleri fluorescent
antisera. One still cannot exclude the possibility that
some alteration of antigens occurred, possibly related
to fixation of tissues, deparaffinization, or time from
death until autopsy. To date, all isolates of Naegleria
obtained from cases of PAM throughout the world
have shown remarkable antigenic homology. Since
cross reactivity between non-pathogenic N. gruberi and
pathogenic N.fowleri is incomplete, it is certainly
possible, at least on an immunologic basis, that the
organism still could be a species of Naegleria other than
N.fowleri, either N. gruberi or an as yet undescribed
species [34, 36].
Thus, the organism responsible for this patient's
death was atypical of both Naegleria and Acanthamoeba, but was a free-living ameba belonging to an
unidentified genus. It may be another species of
Vahlkarnpfia (other than V. avara) as suggested by the
most reliable characteristics available for study, viz.,
the frequent mitosis and division patterns.
Although the responsible pathogen was not precisely identified in this instance, this case is important
because it illustrates that serious diseases may be
produced in humans (and probably in many other
animals) by free-living ameba other than Naegleria,
Hartmannella, Acanthamoeba, and Vahlkampfia. It
emphasizes that the variety of free-living ameba capable of producing disease is as yet undefined, and that it
may be considerable [5, 6]. These organisms are numerous, varied and ubiquitous in nature [15, 22]; they
are distributed world-wide; they multiply rapidly and
appear in a variety of forms; and some species either
prefer or readily invade mammallian tissues. Thus, the
public health implications are obvious, and the scope of
this problem needs to be defined.

Acknowledgements.The authors wish to give special thanks to Dr.
John Moossy,and Dr. Ann Stevensfor their suggestionsand criticism
during the preparation of this manuscript; and Mss. Janet Morrow
and Agnes Zachoszczfor their secretarial assistance.

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Received September 7, 1979/Accepted November 8, 1979