Acta Neuropathologica

Acta Neuropathol (Berl) 51, 85-91 (1980)

9 Springer-Verlag 1980

Original Works Granulomatous Amebic Encephalitis Presenting as a Cerebral Mass Lesion A. Julio Martinez 1, Carlos A. Garcia 2, Meredith Halks-Miller 3, and Rafael Arce-Vela 4 1 2 3 4

Presbyterian-University Hospital, Division of Neuropathology, De Soto at O'Hara Streets, Pittsburgh, PA 15213, USA Louisiana State University Medical Center, New Orleans, LA 70112, USA University of California, San Francisco, School of Medicine, San Francisco, CA 94143, USA Universidad Nacional Federico Villareal (UNFV) y Hospital Central No. 1. SSP, Lima, Per/~

Summary. Clinical and brain biopsy or a u t o p s y findings in six patients with G r a n u l o m a t o u s A m e b i c Encephalitis ( G A E ) due to Acanthamoeba sp. were characterized by focal neurological symptoms, increased intracranial pressure, and focal neuroradiological findings. Craniotomies were performed because o f the diagnostic possibility o f a mass lesion such as a brain t u m o r or abscess. In four patients, frozen sections demonstrated free-living amebas. G A E m a y present as an acute or subacute intracerebral mass lesion with signs and s y m p t o m s o f focal brain disease and should be differentiated f r o m viral, bacterial, fungal, and other protozoal encephalitides. Key words: Acanthamoeba - Amebic encephalitis Corticosteroids - Free-living amebas - I m m u n o suppression - Intracranial mass - Naegleria

Central Nervous System (CNS) disease due to freeliving or amphizoic amebas includes Primary A m e b i c Meningoencephalitis ( P A M ) due to Naegleria fowleri [4, 27, 34] and G r a n u l o m a t o u s A m e b i c Encephalitis ( G A E ) due to Acanthamoeba sp. [2, 8, 10, 12, 1 4 - 18, 24, 25, 31, 32, 35]. P A M is a fulminant, diffuse, hemorrhagic, and necrotizing meningoencephalitis usually f o u n d in young, healthy individuals with a recent history o f water sport activities. G A E refers to an illness with unique clinicopathological characteristics which usually occurs in debilitated and chronically ill individuals, some o f them under immunosuppressive therapy. G A E is usually manifested by focal neurological deficits, signs o f increased intracranial pressure, and neuroradiographic features suggestive o f an expanding intracranial mass [12, 18, 25]. Few reports o f G A E have stressed the focal nature o f the C N S lesions. Offprint requests to: A. Julio Martinez, MD (address see above)

This report describes six patients with G A E who had brain biopsies. In four o f them, frozen sections were diagnostic.

Material and Methods The findings reported here are based on the review of six cases of GAE due to Acanthamoebasp. In four published cases [12, 18, 24], the reported clinical, histopathological description and illustrations of the CNS lesions leave no doubt that the patients had GAE. In addition, immunoperoxidase and immunofluorescent antibody techniques had confirmed the free-living amebic etiology. In addition, two new cases of GAE are added to this series (Cases nos. 5 and 6). Case no. 2 [25] was previously reported [34] as an example of PAM due to Naegleriafowleri, but has been reclassified as an example of GAE due Acanthamoeba castellanii based on IFAT, ultrastructural studies and histopathological features [16, 25]. Light microscopic studies were done from tissues fixed in 10 % buffered formalin, embedded in paraffin and the sections stained with H&E, PAS-H, and Gomori Methenamine Silver. Electron microscopic studies were done in two cases (Cases nos. 2 and 3) from tissues fixed in formalin, deparaffinized and embedded in plastic, following published electron microscopy techniques [10]. Immunofluorescent antibody techniques (IFAT) [38] were performed through the kindness of Drs. Eddy Willaert, William P. Stamm, Johan de Jonckheere, and Clyde G. Culbertson.

Results Epidemiological and Clinical Aspects The patients ranged in age f r o m 6 - 3 2 years. There were five females and one male. Five were white and one black. The clinical duration o f their C N S illnesses ranged f r o m 18 to a b o u t 120 days (Table 1). Clinical s y m p t o m s were those associated with severe meningeal irritation, increased intracranial pressure, and encephalitis: headache, nausea, vomiting, fever, nuchal rigidity, somnolence, dizziness, seizures, aphasia, facial nerve palsy, hemiparesis, cerebellar ataxia, confusion, and hallucinations progressing to c o m a and death (Table 2). T w o patients presented initially with a

0001-6322/80/0051/0085/$1.40

86

Acta Neuropathol (Berl) 51 (1980)

Table 1. Associated disorders and predisposing factors Case no. (Ref. no.)

Age/sex/race year

Clinical course (days) State (USA) or country

Underlying disease or associated disorders

Predisposing factors

Agents

1 [181

6 F 1956

W

~120 Arizona

Skin ulcer (scalp)

Antibiotics

A. culbertson?

2 [16, 25, 34]

7 M 1972

B

~30 Texas

?

Corticosteroids Antibiotics

A. castellanii b

3 [25]

26 F 1972

W

18 Venezuela

Liver disease

Pregnancy (near term) Corticosteroids (terminal)

A. culbertsoni b

4 [121

24 F 1977

W

270(skin) ~ 90 (CNS) Mexican, living' in California

Skin ulcer (arm)

Pregnancy (2 months) Antibiotics Corticosteroids (terminal)

A. astronyxis b

5 (this report)

32 F 1977

W

43 Louisiana

Pneumonitis

Corticosteroids

A. castellanii b

6 (this report)

9 F 1977

W

60 Chincha (300 km south of Lima, Peril)

?

Head injury with skin lesion on scalp. Antibiotics

A. castellanii b

by immunoperoxidase technique b by indirect immunofluorescence antibody technique

chronic ulceration or abrasion of the skin. In one patient, the skin lesion preceded the CNS symptoms by 6 months (Table 1), Craniotomies were performed for brain biopsy in all 6 patients. In four of them, frozen sections of brain biopsies were diagnostic of GAE. All patients had received either systemic corticosteroids or antibiotics prior to development of neurological symptoms. In some cases, however, these agents had been given for a short time or terminally (Table 1). One patient was a pregnant woman near term without evidence of previous illness, but with probable liver dysfunction (liver function tests were abnormally high). Another patient became pregnant one month after the manifestation of CNS symptoms and died 2 months later. Two patients had no known associated disorder or underlying disease. There was no history of water-related sport activities in any patient (Table 2). Two patients were natives of tropical South American countries (Peril and Venezuela); one from Mexico, living in California, and the others were from Southern United States (Arizona, Louisiana, and Texas). Possible pre-disposing factors included: Corticosteroid and broad spectrum antibiotics; pregnancy in two patients; and chronic dermatologic disorders in two patients. Two patients were apparently normal. The findings in the CSF in five cases were a pleocytosis, predominantly lymphocytic from 7 2 -

98 ~, although segmented neutrophils and red blood cells were present. The cell count ranged between 66 and 204 cells per cubic millimeter. The CSF protein average was 80 mg ~ in four of the cases. The CSF glucose in four patients was at the borderline of normal, ranging between 41 and 63 mg ~o (average, 50 mg ~o); and not as low as may be encountered in pyogenic infections.

HistopathoIogy of the Inflammatory Lesions (Table 2) Although the lesions of GAE due to Acanthamoeba sp. in the CNS were similar in all the cases, they varied somewhat from case to case in location, extent, cellular elements, trophozoites, and the number of cysts. Generally, the lesions were most numerous in the cerebellum and brainstem; often the most posterior aspects of the basal ganglia were severely affected, and m o r e rarely, the anterior portion of the cerebral hemispheres. There was usually a focal, chronic or subacute leptomeningitis, near or close to the main lesions. The parenchymal !esions in these cases were all necrotizing granulomatous subacute and chronic encephalitides. Occasionally, there was a small hemorrhagic component in which trophozoites and cysts were found sometimes engulfed by giant celIs (Fig. 3b). Numerous amebic trophozoites and few cysts were occasionally present in the perivascular spaces. Clusters of trophozoites were seen without an inflammatory response. Astrocytosis was usually modest. Sometimes

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

87

Table 2. Epidemiological, clinical features and pathological findings Case no.

Clinical features

1

Oct. 1955: Jan. 1956: March: April: July:

Scalp lesion, right parietooccipital area. Watery fluid exuded from area. Seizures First craniotomy Second craniotomy; left hemiplegia and aphasia.

May

Headache with seizures; left hemiparesis; ataxia; vomiting fever and stiff neck. Suboccipital craniotomy

Autopsy:

1972:

June; 3

Pathological findings Brain biopsy: Autopsy:

Brain biopsy:

Necrotizing chronic granulomatous encephalitis with giant cells and angiitis. Encephalomalacia, right occipital with amebic trophozoites and cysts.

Subacute leptomeningitis and necrotizing granulomatous amebic encephalitis. Granulomas in kidney. Trophozoites in anterior chamber of eye.

Nov.

1972:

Fever for 2 weeks. Headache; seizures; left hemiplegia; coma.

Brain biopsy: Autopsy:

Necrotizing granulomatous encephalitis. Encephalomalacia, fronto-temporal and occipital lobes, basal ganglia, cerebellum, and brainstem with trophozoites and cysts. Granulomas in liver, spleen and myometrium.

Dec.

1976:

Right deltoid area pustule with ulceration. Lesion progressed in 6 months to a 10 x 6 cm noncaseating granuloma. Vomiting and headache. CAT scan (Fig. 1 a) showed two enhancing lesions in right parietal and posterior frontal. Craniotomy

Brain biopsy:

Granulomatous amebic encephalitis (Fig. I b). Encephalomalacia, parietooccipital lobes, basal ganglia, cerebellum and brainstem. Trophozoites in: CNS, adrenals, thyroid, skin, lymph nodes and breast.

Feb.

1977:

Nov.

1976:

Jan.

1977:

Sept. Nov.

I977: 1977:

Autopsy:

Fever with blurred vision, headache, nausea and vomiting for 3 weeks. CAT Scan : Area of decreased density, right posterior parietal and occipital. Two craniotomies

Brain biopsy:

Trauma to head. Dizziness and numbness of left arm. Seizures. Left hemiparesis. Right carotid angiogram (Fig. 3a): Avascular lesion with mass effect. Craniotomy

Brain biopsy:

Autopsy:

Autopsy:

Necrotizing granulomatous amebic encephalitis (Fig. 2a). Encephalomalacia, right posterior parietal and occipital lobes (Fig. 2b), basal ganglia and cerebellum. Amebic "mycotic" aneurysm.Trophozoites and cysts. Granulomatous amebic encephalitis with fibrinoid necrosis and angiitis. Amebic trophozoites (Fig. 3b) but no cysts were found. Not performed

Fig. 1. a CT scan of Case no. 4. There are enhancing lesions in the right parietal and posterior frontal lobes. b Grannlomatous amebic encephalitis. An amebic trophozoite is seen (arrow). Brain biopsy. Giemsa, • 500

88

Acta Neuropathol (Berl) 51 (1980)

Fig. 2. a Granulomatous amebic encephalitis. An amebic trophozoite is seen (arrow). Brain biopsy. H.-E. x 460. h Horizontal section of cerebral hemispheres of Case no. 5. There are hemorrhagic necrotizing lesions on the mesial surfaces of the right and left occipital lobes, and one in the left posterior frontal. The biopsy was taken from the right side (arrow)

Fig. 3. a Right common carotid angiogram of Case no. 6. Anteroposterior view: There is an avascular lesion in the right parietal lobe with mass effect and square shift (arrow) of the anterior cerebral arteries. b Multinucleated giant cell with an amebic trophozoite in the center (arrow). Brain biopsy. H.-E. x 520

"microglial" nodules were observed in the CNS parenchyma. In most cases, there was also a severe angiitis associated with fibrinoid necrosis in some arteries. There was perivascular cuffing by lymphocytes, a few plasma cells, and macrophages and a variable number of eosinophils. Panarteritis or panangiitis with organizing thrombi containing multinucleated giant cells were seen. Amebic trophozoites were present throughout the vascular wall and occasional giant cell and/or trophozoites were also found within the lumens of thrombosed vessels. Discussion

Granulomatous Amebic Encephalitis (GAE) due to Acanthamoeba sp. is of world-wide distribution [28]. It

is of particular interest in that it produces severe and localized necrosis with edema and consequently may mimic other conditions such as cerebral tumor or a brain abscess. The appearance of focal neurological signs and the demonstration of a space occupying avascular mass by carotid angiography with vascular or ventricular shifts in a CAT scan in a patient with encephalitis should alert the neurologist and neurosurgeon to the possibility of GAE. In a recent report, one of us (AJM) has stressed the unique, focal expanding nature of the brain involvement with focal areas of necrotizing hemorrhagic encephalitis [24]. The signs indicative of unilateral space occupying mass are so outstanding that brain tumor or abscess are the primary diagnostic consideration. The possibility of a focal expanding mass lesion is further

A. J. Martinez et al. : GranulomatousAmebic Encephalitis supported in certain cases by the results of ancillary studies, such as EEG, CAT scan, and angiography. GAE has been found in chronically ill, debilitated individuals, some of them under immunosuppressive therapy. Some of the primary non-neurologic symptoms may have been due to the same protozoa, even before receiving systemic corticosteroids and antibiotic therapy. Craniotomy and brain biopsy may be required in order to exclude other surgically treatable lesions like herpes simplex meningoencephalitis, to establish the diagnosis and differentiate viral, fungal or protozoal encephalitides from other cerebral masses like abscesses or neoplasms. Other diagnostic procedures in such lesions are usually non-specific. EEG tracings may show a distinction from a cerebral abscess. The neuroradiological findings provide a guide to the clinical neurologist and to the neurosurgeon as to the character of the lesion, and its site and distribution. GAE should enter into the clinical differential diagnosis of cerebral expanding masses. The isolation and identification of the protozoa from the CNS provides the only specific diagnosis [27]. The importance of an early biopsy is stressed since possible therapeutic success diminishes with time after onset of the disease. The examination of CSF may be of diagnostic value; however, the lumbar puncture might be contra-indicated because of signs of increased intracranial pressure. It appears that Acanthamoeba sp. has a predilection for attacking vascular walls, thus leading to necrotizing arteritis and sometimes aneurysm formation with thrombosis or hemorrhage. It seems logical to assume that the mechanism of aneurysmal formation is amebic invasion from within the artery. Virulence of the infective organism determines whether meningitis, encephalitis or local aneurysm will occur [1]. The majority of cases of bacterial "mycotic" aneurysm have been associated with bacterial endocarditis, or septicemia which may result when infected emboli lodge in a cerebral vessel [1, 13]. Reasonable sources for blood stream infection may be found in the majority of cases of GAE, so the entry into the CNS by the amebas seems to be through hematogenous route, from a portal of entry possibly the skin or lungs [28]. Histologic examination of the inflammatory component around the cysts reveals that the majority of the cells are mainly lymphocytes and plasma cells indicating abundant antibody production. Little chronic or subacute inflammatory activity is observed around some trophozoites. The differences in cellular composition between PAM and GAE suggests a different pathogenesis of these two diseases. The means by which the amebas gain access to the CNS may be speculated as

89 follows : The protozoa or infected emboli lodge on the intimal surface of the artery, affecting the vasa vasorum. The septic emboli bring the infected material directly in contact with the endothelial lining and produced the disruption, dilatation, and weakness of the vessel wall with the possible formation of an aneurysm. The wall of the vessel may become infected from the inside of the lumen or from outside the vessel. Infections from outside usually are the result of the extension of an acute or chronic process from an adjacent structure. Acanthamoeba sp. may be present in the human nasopharynx as part of the normal flora [6, 33], or in the environment [19, 20]. Free-living amebas have been isolated from samples of human origin [36, 37] or from contamination of tissue cultures [29]. Wang and Feldman, using tissue cultures, have isolated strains of Acanthamoeba (Hartmannella) from human throats [36, 37]. Callicott et al. [4] isolated an ameba identified as A. astronyxis from the CSF of a patient with an "aseptic" meningitis that remitted spontaneously. A contaminant cannot be ruled out. In this case, the CSF was not directly examined for motile amebas. Up to the present time, Acanthamoeba sp. has not been identified in CSF samples. Abscesses [31 and infections of ears [21] and eyes, with ulceration of the cornea or amebic keratitis and endophthalmitis have been reported [16, 22, 30]. Sko~il et al. [33] and Cerva et al. [6] obtained isolates of free-living amebas from nasopharyngeal swabs in healthy soldiers. They obtained a higher proportion of positive cultures from subjects with nasopharyngeal symptoms. Dyner [11] sampled the urine from patients on immunosuppressive drugs and steroids and found free-living amebas in about 7 ~. Some organisms, which under normal circumstances would be considered of low or little pathogenicity, ,could in cases of severe debilitating diseases or injury become "opportunists", gain access to the bloodstream and cause a fatal infection [ 7 - 9 , 23, 26]. It has been demonstrated in experimental animals that the administration of corticosteroids and broad spectrum antibiotics suppresses host defenses and depresses host immunity allowing for the establishment of amebic encephalitis and pneumonitis [7, 9, 23, 26]; and therefore, steroids may be a contraindication in infected patients. This presents a major problem in that definitive diagnosis may, in some cases, only be obtained after craniotomy for which steroids are routinely used. The frequent use of corticosteroids and broad spectrum antibiotics inhibits or suppresses normal bacterial flora allowing colonization and overgrowth of "opportunist" fungi. Some damaging effects of the most superficial layers of the mucous membranes (like the gastro-intestinal tract or in the ocular tissues) may

90 be p r o d u c e d b y antibiotics~ f a c i l i t a t i n g the p e n e t r a t i o n of opportunistic pathogens. T h e t r e a t m e n t w i t h s u l f a d i a z i n e is p r o b a b l y ineffective in h u m a n s ; a l t h o u g h this m a y b e a r e f l e c t i o n o f the i n a d e q u a c y o f t h e h o s t ' s i m p a i r e d i m m u n e system. S u l f a d i a z i n e is active in p r e v e n t i o n a n d t r e a t m e n t o f t h e disease in m i c e i n f e c t e d i n t r a n a s a l l y w i t h A c a n t h a m o e b a sp,, b u t has little o r n o effect o n s i m i l a r Naegleria f o w l e r i i n f e c t i o n [91. In vitro 5-fluorocytosine and hydroxystilbamidine h a v e b e e n u s e d w i t h a d d i t i o n a l p r o t e c t i v e effect in e x p e r i m e n t a l a n i m a l i n f e c t i o n [5]. T h e r e are n o effective t r e a t m e n t s a v a i l a b l e at this time. N e u r o s u r g e o n s a n d n e u r o l o g i s t s t a k i n g care o f p a t i e n t s w i t h the disease p r o d u c e d b y a m p h i z o i c or free-living a m e b a s s h o u l d c o n t a c t T h e C e n t e r for D i s e a s e C o n t r o l in A t l a n t a , G e o r g i a ( U S A ) [ d u r i n g t h e d a y , t h e n u m b e r is (404) 329-3311 ; a n d at n i g h t a n d h o l i d a y s , t h e n u m b e r is (404) 329-3644]. A w a r e n e s s o f the p o s s i b l e o p p o r t u n i s t i c n a t u r e o f A c a n t h a m o e b a sp. i n f e c t i o n w i t h its u n e x p e c t e d dissem i n a t i o n w i t h o u t t h e p r e s e n c e o f clinical s y m p t o m s o r e p i d e m i o l o g i c a l clue m a y a l l o w earlier d i a g n o s i s o f f u t u r e cases, a d v a n c i n g the s t u d y o f p a t h o g e n e s i s a n d t h e r a p y o f this n e w l y r e c o g n i z e d p r o t o z o a l i n f e c t i o n . Acknowledgements. The authors wish to give special thanks to Dr. C. Sotelo-Avila for supplying Case no. 2; Dr. Jorge Garcia-Tamayo for supplying Case no. 3 ; and Drs. Juan Gutierrez Manay and Carmen Asato-Higa for supplying Case no. 6; to Dr. John Moossy for valuable suggestions during the preparation of this manuscript; to Drs. Clyde Culbertson, Eddy Willaert, W. P. Stamm, and J. DeJonckheere for performing indirect immunofluorescent antibody and immunoperoxidase techniques; and to Ms. Janet Morrow and Ms. Agnes Zachoszcz for secretarial assistance.

References

1. Bell W, Butler E (1968) Cerebral mycotic aneurysms in children. Two case reports. Neurology (Minneap) 18:81 - 86 2. Bhagwandeen SB, Carter RR, Naik KG, Levitt D (1975) A case of Hartmanellid amebic meningoencephalitis in Zambia. Am J Clin Pathol 63:483--492 3. Borochovitz D, Martinez AJ, Patterson TG (1980) Osteomyelitis of bone graft of mandible with Acanthamoeba castellanii infection. Hum Pathol (in press) 4. Callicott JH, Nelson EC, Jones MM, dos Santos JG, Utz JP, Duma RJ, Morrison JV (1968) Meningoencephalitis due to pathogenic free-living amoebae. JAMA 206: 579- 582 5. Casemore DP (1970) Sensitivity of Hartmannella (Acanthamoeba) to 5-fluoro cytosine, hydroxystilbamidine and other substances. J Clin Pathol 23:649-652 6. Cerva L, Serbus C, Sko~il V (1973) Isolation of Limax amoeba from the nasal mucosa of man. Folia Parasitol (Praha) 20:97103 7. Culbertson CG, Smith J, Cohen H, Minner J (1959) Experimental infection in mice and monkeys by Acanthamoeba. Am J Pathol 35:185 - 1 9 7 8. Culbertson CG ( 1 9 6 1 ) Pathogenic Acanthamoeba (Hartmannella). Am J Clin Pathol 35:195-202

Acta Neuropathol (Berl) 51 (1980) 9. Culbertson CG (1971) The pathogenicity of soil amebas. Ann Rev Microbiol 25: 231 - 254 10. Duma RJ, Helwig WB, Martinez AJ (1978) Meningoencephalitis and brain abscess due to free-living amoeba. Ann Intern Med 88:468-473 11. Dyner E (1974) Les immunosuppresseurs et les amibes de l'eau. Ann Soc Belg Med Trop 54:405-408 12. Gullett J, Mills J, Hadley K, Podemski B, Pitts L, Gelber R (1979) Disseminated granulomatous Acanthamoeba infection presenting as an unusual skin lesion. Am J Med 67:891- 896 13. Heidelberger KP, Layton WM, Fisher RG (1968) Multiple cerebral mycotic aneurysm complicating post-traumatic Pseudomonas meningitis. J Neurosurg 29 : 631 - 635 14. Hoffmann EO, Garc[a C, Lunseth J, McGary P, Coover J (1978) A case of primary amebic meningoencephalitis. Light and electron microscopy and immunohistologic studies. Am J Trop Med Hyg 27:29-38 15. Jager BV, Stamm WP (1972) Brain abscesses caused by freeliving amoeba probably of the genus Hartmannella in a patient with Hodgkin's Disease. Lancet II:1343-1345 16. Jones DB, Visvesvara GS, Robinson M (1975) Ac~hjhamoebi~ polyphaga keratitis and Acanthamoeba uveitis associated with fatal meningoencephalitis. Trans Ophthalmol Soc U K 95 : 221 232 17. Kenney M (1971) The micro-kolmer complement fixation test in routine screening for soil ameba infection. Health Lab Sci 8:5-10 18. Kernohan JW, Magath TB, Schloss GT (1960) Granuloma of brain probably due to Endolimax williamsi (Iodamoeba bi~tschlii). Arch Pathol 70: 576- 580 19. Kingston D, Warhurst DC (1969) Isolation of ameba from the air. J Med Microbiol 2 : 2 7 - 3 6 20. Lawande RV, Abraham SN, John I, Egler LJ (1979) Recovery of soil amebas from nasal passages of children during the dusty Harmattan Period in Zaria. Am J Clin Pathol 71:201203 21. Lengy J, Jakovlzevich R, Tolis B (1971) Recovery of a Hartmannelloid ameba from a purulent ear discharge. Trop Dis Bull 68:818 22. Lund O, Stefani FH, Dechant W (1978) Amoebic keratitis. A clinicopathological case report. Br J Ophthalmol 62:373-375 23. Markowitz SM, Sobieski T, Martinez AJ, Duma RJ (1978) Experimental Acanthamoeba infections in mice pretreated with methylprednisolone or tetracycline. Am J Pathol 92:733- 741 24. Martinez AJ, Sotelo-Avila C, Alcal~t H, Willaert E (1980) Granulomatous encephalitis, intracranial arteritis and mycotic aneurysm due to a free-living ameba. Acta Neuropathol (Bed) 49:7-12 25. Martinez AJ, Sotelo-Avila G, Garcia-Tamayo J, Takano-Mor6n J, Willaert E, Stamm WP (1977) Meningoencephalitis due to Acanthamoeba sp. : Pathogenesis and clinicopathological study. Acta Neuropathol (Berl) 37:183 - 191 26. Martinez AJ, Markowitz SM, Duma RJ (1975) Experimental pneumonitis and encephalitis caused by Acanthamoeba in mice: Pathogenesis and ultrastructural features. J Infect Dis 1313:692-699 27. Marfinez AJ, dos Santos JG, Nelson EC, Stamm WP, Willaert E (1977) Primary amebic meningoencephalitis. In: Sommers SC, Rosen PP (eds) Pathology annual, vol 12, part 2. AppletonCentury-Crofts, New York, pp 225-255 28. Martinez AJ (1980) Is A canthamoeba encephalitis an opportunistic infection? Neurology (Minneap) 30: 567- 574 29. Moore AE, Hlinka J (1968) Hartmannella sp. (Acanthamoeba) as a tissue culture contaminant. J Natl Cancer Inst 40: 569- 581 30. Nagington J, Watson PG, Playfair TJ, McGill J, Jones BR, McG Steele AD (1974) Amoebic infection of the eye. Lancet II : 15371540

A. J. Martinez et al. : Granulomatous Amebic Encephalitis 31. Ringsted J, Jager BV, Suk D, Visvesvara GS (1976) Probable Acanthamoeba meningoencephalitis in a Korean child. Am J Clin Pathol 66: 7 2 3 - 730 32. Robert VB, Rorke LB (1973) Primary amebic encephalitis probably from Acanthamoeba. Ann Intern Med 79:174--179 33. Sko~il V, Cerva L, Serbus C (1970) Epidemiological study of amoebas of the Limax group in military communities. First report. J Hyg Epidemiol Microbiol Immunol (Praha) 14: 61 - 66 34. Sotelo-AviIa C, Taylor EM, Ewing CW (1974) Primary amebic meningoencephaiitis in a healthy 7-year-old boy. J Pediatr 85:131-136 35. Takano-Mor6n J, Cabrera J, Franco F, Martinot C (1976) Meningoencephalitis Amibiana primaria subaguda. Patologia (Mexico City) 14:287-297

Note Added in Proof

Since the submission of this paper, two additional cases of Amebic Encephalitis (GAE) due to Acanthamoeba sp. were seen in Pittsburgh in which brain biopsies were diagnostic: 1. A 21/2 year old white boy originally from Texas died in the Children's Hospital of Pittsburgh after 71/2 months clinical course (Wessel HB, et al. (1980) MMWR 29:117; Wessel HB, et al. (1980) Neurology (Minneap) 30: 442). 2. A 38 year old white man, a renal transplant recipient, immunosuppressed since October 1977 died after a stormy clinical course of about 6 weeks with associated pulmonary cytomegalovirus and Candida albicans infections, and multiple subcutaneous amebic lesions.

91 36. Wang SS, Feldman HA (1961) Occurrence of Acanthamoeba in tissue cultures inoculated with human pharyngeal swab. Antimicrobiol Agents Chemother 1 : 5 0 - 53 37. Wang SS, Feldman HA (1967) Isolation of Hartmannella species from human throats. N Engl J Med 277:1174--1l 79 38. Willaert E, Stevens AR, Healy G R (1978) Retrospective identification of Acanthamoeba culbertsoni in a case of amoebic meningoencephalitis. J Clin Pathol 31:717-720

Received March 14, 1980/Accepted April 24, 1980