5399
Infection of the Central Nervous System Due to Acanthamoeba A. Julio Martinez
From the Pathology Department (Neuropathology), PresbyterianUniversity Hospital, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
It is wellestablished that Acanthamoeba castellanii, Acanthamoeba culbertsoni, Acanthamoeba polyphaga, and probably other species of free-living ~meb~ ar~ virul~nt op~ortunists ~pable
For many years Entamoeba histolytica was thought to be the only ameba pathogenic for mammals. In the last few years, however, it has become apparent that several species of freeliving amebas can infect human beings and animals and that death may result from such infections [1]. Two main genera of free-living amebas have been found to infect humans: Naegleria and Acanthamoeba. Naegleria fowleri may produce an acutely fatal infection of the CNS that is known as primary amebic meningoencephalitis (PAM) and resembles fulminant bacterial meningitis. PAM usually occurs in healthy young individuals with a history of either swimming in or recent nasal contact with contaminated water [2~. Species of Acanthamoeba may cause a subacute or chrome CNS infection known as granulomatous amebic encephalitis (GAE) [3]. In addition, acanthamoebas may produce acanthamoebic keratitis [4], pneumonitis, and subacute granulomatous dermatitis. CNS infection due to Acanthamoeba species usually occurs in patients who have impaired defense mechanisms or who are ill and debilitated and, in many cases, are being treated with immunosuppressive drugs or broad-spectrum antibiotics. The infections produced by Acanthamoeba species are considered to be opportunistic [3]. Considerable progress has been made in the recognition of the symptoms and the histomorphologic features of PAM and GAE, and more cases are likely to be reported in the future as the medical community becomes more aware of procedures for isolating, cultivating, and identifying the causative organisms.
Reprints and correspondence: Dr. A. 1. Martinez, Division ofN.europathoiogy, Presbyterian-University Hospital, Pittsburgh, Penns~lvama 15213. This work was supported in part by the Pathology Education and Research Foundation of the Department of Pathology, University of Pittsburgh. Reviews of Infectious Diseases 1991;13(8uppl5):8399-402 © 1991 by The University of Chicago. All rights reserved. 0162-0886/91/1302-0070$02.00
Epidemiologic Aspects of GAE The epidemiology of Acanthamoeba species is both complex and intriguing. Acanthamoebas are inhabitants of soil, water, sewage and wastes, thermally polluted waters, cooling towers, air-conditioning filters, and fecal material from birds, reptiles, and mammals [4-6]. The air and dust may carry amebic cysts. Infections have been observed in humans, cattle, reptiles, dogs, and fish. The increased interest in these amebas bas led to descriptions of new species isolated from aquariums, swimming pools, throat swabs, human stools, contaminated cell cultures, marine sediments, and contact lenses [7-13]. Acanthamoeba castellanii, Acanthamoeba culbertsoni, Acanthamoeba polyphaga, and Acanthamoeba palestinensis are the better-known species. Some newly identified species include Acanthamoeba royreba, Acanthamoeba lenticulata, Acanthamoeba hatchetti, Acanthamoeba griffini, and Acanthamoeba triangularis, but there are still several unidentified species that probably are responsible for infections of the CNS, cornea, respiratory tract, and/or skin [14, 15]. Experimental studies in several laboratories have shown that some acanthamoebas may ingest the bacteria responsible for Legionnaire's disease and act as aerosol carriers [6]. Other studies of factory workers who have become ill with "humidifier" fever or with respiratory symptoms such as hypersensitivity pneumonitis have shown that acanthamoebas can be present in water from cooling towers and in air filters [13]. So far, no animal reservoir of free-living amebas has been demonstrate as a carrier.
eNS Involvement by Acanthamoeba Species The route of invasion and penetration of acanthamoebas into the eNS in cases of GAE appears to be hematogenous, probably with a primary focus in the lower respiratory tract or
Downloaded from http://cid.oxfordjournals.org/ at UQ Library on August 15, 2015
of producing disease in humans and animals. Human infections involving braIn: eyes,. skin, and lungs have been reported from all continents. ~entr~l nerv~~s sys~m ~~NS) infection due to Acanthamoeba species usually occurs in chromcally ill, debilitated individuals, some of them receiving immunosuppressive therapy or taking broad-spectrum antibiotics. T~e disease ~?S a protracted, insidious clinical course and is known as granulo~atous ameb~c encephaliti». Histopathologically, Acanthamoeba species may produce a m~tifocal, c~omc, or subacute granulomatous encephalitis, with trophozoites and cysts present In C~S les~ons. The portal of entry of the amebas into the CNS is probably the respiratory tract or a skin lesion, and the organisms reach the CNS by hematogenous spread. As of 1 January 1989,about SO cases of granulomatous amebic encephalitis had been reported worldwide, 27 in the United States alone.
S400
Martinez
through an ulceration of the skin. In experimental animals the olfactory neuroepithelium has also been found to be a possible portal of entry [2]. The incubation period of GAE is unknown but is probably longer than 10 days [2]. The ability of acanthamoebas to produce necrosis of brain tissue is probably due to an enzymatic action induced by lysosomal hydrolases and a phospholipase that can degrade phospholipids of the myelin sheaths [14].
Clinical Manifestations of GAE
Pathologic Features of GAE Macroscopic appearance ofinfected CNS tissues. In GAE the cerebral hemispheres show moderate edema. The leptomeninges may contain a moderate amount of purulent exudate, with minimal cloudiness over the most affected cortical areas. In the less affected regions, the leptomeninges are transparent [2]. There are foci of encephalomalacia and softening associated with discrete or confluent areas of hemorrhagic necrosis in occipital, parietal, temporal, or-less often-frontal lobes. The lesions are usually multifocal, located in the posterior fossa structures, midbrain, thalamus, brainstem, corpus callosum, and cerebellum. The cervical portion of the spinal cord is occasionally affected. Bilateral uncal notching and
cerebellar tonsillar herniations may be prominent features. The olfactory bulbs and base of the frontal lobes are usually spared [2]. Microscopic findings in infected CNS tissues. The lesions of GAE due to Acanthamoeba species in the CNS may vary from case to case in location, extent, composition of cellular elements, and density of trophozoites and cysts. As a rule, the lesions are most numerous in the basal ganglia, midbrain, brainstem, and cerebral hemispheres [2]. Usually there is a focal, rather modest, chronic or subacute leptomeningitis, most of which is close to parenchymal lesions. The characteristic lesion in the CNS parenchyma is a necrotizing subacute or chronic granulomatous encephalitis in which trophozoites and cysts may be found in association with "giant cells;' some of them engulfing amebas. The amebic trophozoites and cystsmay be present in large numbers, mainly in perivascular spaces of the affected areas. Sometimes clusters of trophozoites are seen without an accompanying inflammatory response. Gliosis around necrotic areas is usually modest. Phagocytic nodules may be observed. Foci of recent hemorrhages may be seen, usually within the necrotic areas. In some cases there is severe angiitis (panarteritis), with perivascular cuffing by lymphocytes, some plasma cells, few macrophages, and rare eosinophils.Fibrinoid necrosis and thrombosis constitute an interesting feature in some instances. Amebic trophozoites and cysts may be found piercing the vascular wall. In some cases, particularly in patients with AIDS, the absence of a granulomatous reaction indicates severely compromised host defense mechanisms. At postmortem, involvement of viscera other than the CNS may be found, suggestinga premortem hematogenous dissemination of trophozoites and cysts. The organs involved may be subcutaneous tissue and skin, liver, lungs, kidneys, adrenals, pancreas, prostate, lymph nodes, and myometrium [2, 33].
Diagnosis of GAE Laboratory methods, differential diagnosis, direct microscopic diagnosis, and cultures. The CSF findings may be of value in the diagnosis of acanthamoeba infection [34]. Pleocytosis is the rule, with lymphocytic predominance. The opening pressure is slightly elevated; the glucose concentration is usually low and the protein concentration moderately high. Polymorphonuclear leukocyte counts are also elevated but not as high as in PAM due to N. fowleri. The CSF is not as frankly purulent as in bacterial meningitis. Amebic trophozoites may be present in the CSF [32]. An important laboratory procedure for the diagnosis of infections due to free-living amebas is the microscopic examination of CSF. Trophozoites may be found in cases of PAM due to N. fowleri. Motile amebas are sought by placing one or two drops of unstained CSF (which has been centrifuged at low speed) on a glass slide, coverslip, or hanging drop and then using an ordinary light microscope with lowered diaphragm for phase-contrast or dark-field illumination. Amebas
Downloaded from http://cid.oxfordjournals.org/ at UQ Library on August 15, 2015
Symptoms and signs. Clinical symptoms of GAE due to Acanthamoeba species are mainly those of focal or localized encephalopathy with subsequent severe meningeal irritation and encephalitis, i.e., mental status abnormalities and behavioral changes (confusion, irritability, somnolence, hallucinations, and dizziness), seizures, headache, hemiparesis, aphasia, cranial nerve palsies, fever, stiff neck, visual disturbances (diplopia and blurred vision), anorexia, nausea and vomiting, ataxia, papilledema, coma, and death [16-40]. Clinical course: morbidity, mortality, and prognosis. The clinical presentation of GAE is insidious and may mimic that of bacterial leptomeningitis, tuberculous meningitis, or viral encephalitis. Some patients may present initially with respiratory infection or with gastrointestinal symptoms; some with a chronic ulceration of the skin or with skin nodules; and others with symptoms mimicking a space-occupying mass, a brain abscess, or a brain tumor. In the latter cases craniotomies for a brain biopsy and frozen section may unravel the diagnosis. Clinical setting, risk, and predisposing factors. GAE due to Acanthamoeba species may occur in individuals who are pregnant, chronically ill and debilitated, chronically alcoholic, or malnourished. Some patients have underlying diseases such as skin ulcers or skin trauma, lymphomas, diabetes mellitus, cirrhosis of the liver or other hepatic disease, AIDS, gammaglobulinemia, and glucose-6-phosphate dehydrogenase deficiency [2]. Patients at risk are those receiving treatment with steroids, antibiotics, and immunosuppressive drugs (e.g., agents used to prevent organ rejection in transplant recipients).
RID 1991;13 (Suppl 5)
RID 1991;13 (Suppl 5)
CNS Acanthamoeba Infection
differentiation of trophozoites of N. fowleri and Acanthamoeba species in CNS tissue may be difficult by light microscopy, but trophozoites of Acanthamoeba are larger (l5-4~ I'm in diameter). They characteristically contain a centrally placed, dense karyosome or nucleolus that is surrounded by a clear nuclear halo of chromatin and a double nuclear membrane. The nucleus measures "-15-10 I'm in diameter. The cytoplasm is abundant with slender hyaline projections or acanthopodia. Clear and dense vacuoles and dense granules are seen in the cytoplasm. The mean diameter of cystsof Acanthamoeba species is "-115-20 I'm. Diagnosis by electron microscopic techniques. The ultrastructural features of amebic trophozoites and cysts of Acanthamoeba species have been described for postmortem material as well as for axenic cultures and in the experimental disease in animals. Electron microscopic examination of pathogenic acanthamoebas may reveal numerous oval, round, and "dumbbell-shaped" mitochondria. Rough and smooth endoplasmic reticulum is abundant. The Golgi apparatus is prominent. Food vacuoles containing myelin figures are conspicuous. Some empty vacuoles are also seen. The karyosome is composed of dense RNA, and the nuclear chromatin is finely granular and surrounded by a double membrane. During nuclear division the karyosome may divide in two and the nuclear membrane is absent - primarily during the late prophase. The cysts of Acanthamoeba species have a stellate, spherical, or polyhedral shape, with double wrinkled walls or a solid, compact wall. Ostioles and opercula in which excystation takes place may be seen. The cyst walls are laminated and united at the level of opercula.
Treatment of GAE At this time no effectivetreatment is available for CNS acanthamoeba infection. In contrast, ophthalmic infections such as acanthamoeba keratitis have been treated with success in some cases. For example, propamidine isethionate (Brolene), an ophthalmic medication that can be obtained over the counter in England, has been reported to be effective in the treatment of acanthamoeba keratitis. Neomycin also appears to be effective in combination with propamidine isethionate. The efficacy of these drugs in GAE is not known. Sulfadiazine is probably ineffective, perhaps because of the inadequacy of the host's immune system. Ketoconazole appears to be effective both in vitro and in vivo (in experimental animal models) for treatment of CNS infections. Acridine derivatives and paromomycin seem promising in vitro, but the use of these compounds is still in the experimental stage [43, 44].
References 1. Martinez AJ. Free-living amebas: natural history, prevention, pathology, and treatment of disease. Boca Raton, FL: CRC Press, 1985:156 2. Martinez AJ. Free-living amoebas: pathogenic aspects. A review. Protozoology Abstracts 1983;7:293-306
Downloaded from http://cid.oxfordjournals.org/ at UQ Library on August 15, 2015
are refractile and can be seen moving. They must be differentiated from lymphocytes, monocytes, polymorphonuclear leukocytes, and macrophages. Naegleria species are highly motile, with lobopodia erupting rapidly. Motility is one of the most important features to observe. Acanthamoeba species are sluggish and may be confused with macrophages. A few cases of acanthamoeba infection have been diagnosed by microscopic observation of the motile but sluggish organisms [34]. Giemsa- or Wright-stained smears of centrifuged CSF offer the advantage of showing the characteristic morphology of the amebas that differentiates them from lymphocytes, erythrocytes, and polymorphonuclear leukocytes. An abundance of erythrocytes may suggest PAM due to N. fowleri, which is characterized by necrosis and hemorrhage [1]. Cultures can be prepared from the CSF, or from brain tissue, but few cases of GAE have been diagnosed by means of such cultures [34]. Acanthamoeba species are considered synonymous with Hartrnannella by some protozoologists. This group of organisms is characterized by a centrally placed, dense nucleolus that is surrounded by a clear nuclear halo and abundant cytoplasm. Usually gram-stained smears and bacterial cultures are negative. Amebas can easily be seen in tissues stained with hematoxylin and eosin. Cysts are better seen with periodic acid-Schiff hematoxylin or Grocott methenamine silver staining. Retrospective diagnosis of GAE. The great majority of cases of GAE have been diagnosed postmortem by the detection of amebic trophozoites and cysts in sections of CNS tissue embedded in paraffin and stained with hematoxylin and eosin [41]. Immunofluorescent antibody and immunoperoxidase methods. When no cultures have been prepared or isolates obtained, the indirect immunofluorescent antibody test (IFAT) or immunoperoxidase staining (IPAS) of CNS tissue fixed in formalin and embedded in paraffin is valuable for the identification and localization of trophozoites and cysts [42]. The tissues are treated with specific, labeled antiserum. However, there is considerable antigenic overlap between species of the genus Acanthamoeba (A. castellanii, A. culbertsoni, A. polyphaga, and Acanthamoeba astronyxis) and other free-living amebas. The use of IFAT and IPAS may permit more precise identification than is possible by morphologic study of the trophozoites and cysts. In addition, immunodiffusion or immunoelectrophoretic analysis of water-soluble proteins of the amebic trophozoites from cultures may be used for the identification and comparison of free-living strains of ameba. In some cases the IPAS or IFAT is inconclusive and the diagnosis can be reasonably established only by observation of the morphologic features of the trophozoites and cysts and the histopathologic features of the affected CNS tissue. The diagnosis of possible vahlkampfia infection may be suggested on the basis ofbinucleation of some of the trophozoites in the CNS lesions or of negative IFAT and IPAS results in tests for several Acanthamoeba species and N. fowleri. Diagnosis by light microscopic techniques. Morphologic
5401
5402
Martinez
25. Cox EC. Amebic meningoencephalitis caused by Acanthamoeba species in a four month old child. J S C Med Assoc 1980;76:459-62 26. Wessel HB, Hubbard I, Martinez AI, Willaert E. Granulomatous amebic encephalitis (GAE) with prolonged clinical course: CT scan findings, diagnosis by brain biopsy and effect oftreatment [abstract]. Neurology (Minneap) 1980;30:442 27. Gullett I, Mills I, Hadley K, Podemski B, Pitts L, Gelber R. Disseminated granulomatous Acanthamoeba infection presenting as an unusual skin lesion. Am J Med 1979;67:891-6 28. Martinez AI, Sotelo-AvilaC, Alcala H, Willaert E. Granulomatous encephalitis, intracranial arteritis, and mycotic aneurysm due to a freeliving ameba. Acta Neuropathol (Berl) 1980;49:7-12 29. Grunnet ML, Cannon GH, Kushner JP. Fulminant amebic meningoencephalitis due to Acanthamoeba. Neurology (Minneap) 1981;31:174-7 30. Carter RF, Cullity GJ, Ojeda VJ, Silberstein P, Willaert E. A fatal case of meningoencephalitis due to a free-living amoeba of uncertain identity-probably Acanthamoeba sp. Pathology 1981;13:51-68 31. Borochovitz D, Martinez AI, Patterson GT. Osteomyelitis ofa bone graft of the mandible with Acanthamoeba castellaniiinfection. Hum Pathol 1981;12:573-6 32. Cleland PG, Lawande RV, OnyemelukweG, Whittle He. Chronic amebic meningoencephalitis. Arch Neurol 1982;39:56-7 33. Martinez AI. Acanthamoebiasis and immunosuppression. Case report. I Neuropathol Exp Neurol 1982;41:548-57 34. Lalitha MK, Anandi V, Srivastava A, Thomas K, Cherian AM,. Chandi SM. Isolation of Acanthamoeba culbertsoni from a patient with meningitis. I Clin Microbiol 1985;21:666-7 35. Gonzalez MM, Gould E, Dickinson G, Martinez AI, Visvesvara G, Cleary TI, Hensley GT. Acquired immunodeficiency syndrome associated with Acanthamoeba infectionand other opportunistic organisms. Arch Pathol Lab Med 1986;110:749-51 36. Wiley CA, Safrin RE, Davis CE, Lampert PW, Braude AI, Martinez AI, Visvesvara GS. Acanthamoeba meningoencephalitis in a patient with AIDS. I Infect Dis 1987;155:130-3 37. Ofori-Kwakye SK, Sidebottom DG, Herbert J, Fischer EG, Visvesvara GS. Granulomatous brain tumor caused by Acanthamoeba. Case report. I Neurosurg 1986;64:505-9 38. Rutherfoord GS. Amoebic meningo-encephalitis due to a free-living amoeba. S Afr Med J 1986;69:52-5 39. Matson DO, Rouah E, Lee Rf, Armstrong D, Parke IT, Baker C1. Acanthameba meningoencephalitis masquerading as neurocysticercosis. Pediatr Infect Dis J 1988;7:121-4 40. Harwood CR, Rich GE, McAleer R, Cherian G. Isolation of Acanthamoeba from a cerebral abscess. Med I Aust 1988;148:47-9 41. Willaert E, Stevens AR, Healy GR. Retrospective identification of Acanthamoeba culbertsoni in a case of amoebic meningoencephalitis. J Clin Pathol 1978;31:717-20 42. Willaert E, Stevens AR. Indirect immunofluorescent identification of Acanthamoeba causing meningoencephalitis. Pathol BioI 1976;24: 545-7 43. Steven AR, Willaert E. Drug sensitivity and resistance of four Acanthamoeba species. Trans R Soc Trop Med Hyg 1980;74:806-8 44. Osuna A, Rodriguez-Santiago11,Ruiz-Perez L-M, Gamarro F, Castanys S, Giovannangeli G, Galy AM, Galy IP, Soyfer JC, Barbe 1. Antiamebic activity of new acridinic derivatives against Naegleria and Acanthamoeba species in vitro. Chemotherapy 1987;33:18-21
Downloaded from http://cid.oxfordjournals.org/ at UQ Library on August 15, 2015
3. Martinez AI. Is acanthamoebic encephalitis an opportunistic infection? Neurology (New York) 1980;30:567-74 4. Lewis EJ, Sawyer TK. Acanthamoeba tubiashi N. Sp., a new species of fresh-water Amoebida (Acanthamoebidae). Transactions of the American Microscopy Society 1979;98:543-8 5. SawyerTK, Visvesvara GS, Harke EA. Pathogenicamoebas from brackish and ocean sediments, with a description of Acanthamoeba hatchetti, n. sp. Science 1977;196:1324-5 6. RowbothamTI, et al. Preliminary report on the pathogenicityof Legionella pneumophila for freshwater and soil amoebae. J Clin Pathol 1980;33:1179-83 7. Casemore DP. Contamination of virological tissue cultures with a species of free-living soil amoeba. J Clin Pathol 1969;22:254-7 8. Willaert E, Stevens AR, Tyndall RL. Identification of Acanthamoeba culbertsoni from cultured tumor cells. Protistologica 1978;14:319-36 9. Wang SS, Feldman HA. Isolation of Hartmannella species from human throats. N Engl J Med 1967;277:1174-9 10. Visvesvara GS, Mirra SS, Brandt FH, Moss DM, Mathews HM, Martinez AI. Isolation of two strains of Acanthamoeba castellanii from human tissue and their pathogenicity and isoenzyme profiles. J Clin Microbiol 1983;18:1405-12 11. Tyndall RL, Willaert E, Stevens A, Nicholson A. Pathogenic and enzymatic characteristics of Acanthamoeba isolated from cultured tumor cells. Protistologica 1979;15:17-22 12. DeJonckheere JE Pathogenic and nonpathogenic Acanthamoeba spp. in thermally polluted discharges and surface waters. J Protozool 1981;28:56-9 13. Edwards JH, Griffiths AI, Mullins J. Protozoa as sources of antigen in "humidifier fever." Nature 1976;264:438-9 14. Costas M, Griffiths AF. Enzyme composition and the taxonomy of Acanthamoeba. J Protozool 1985;32:604-7 15. Page Fe. Re-definition of the genus Acanthamoeba with descriptions of three species. I Protozool 1967;14:709-24 16. Kernohan J, Magath TB, Schloss GT. Granuloma of brain probably due to Endolimax williansi (Iodamoeba butschliiy. Archives of Pathology 1960;70:576-80 17. Robert VB, Rorke LB. Primary amebic encephalitis, probably from Acanthamoeba. Ann Intern Med 1973;79:174-9 18. Jager BV, Stamm WP. Brain abscesses caused by a free-living amoeba probably of the genus Hartmannella in a patient with Hodgkin's disease. Lancet 1972;2:1343-5 19. Bhagwandeen SB, Carter RF, Naik KG, Levitt D. A case of Hartmannellid amebic meningoencephalitis in Zambia. Am J Clin Pathol 1975;63:483-93 20. Ringsted I, Jager BV, Suk D, Visvesvara GS. Probable Acanthamoeba meningoencephalitis in a Korean child. Am J Clin Pathol 1976; 66:723-30 21. Martinez AI, Sotelo-Avila C, Garcia-Tamayo J, 'Iakano-Moron I, Willaert E, Stamm WP. Meningoencephalitis due to Acanthamoeba SP: pathogenesis and clinico-pathological study. Acta Neuropathol (Berl) 1977;37:183-91 22. Duma RJ, Helwig WB, Martinez AI. Meningoencephalitis and brain abscess due to free-living amoebae. Ann Intern Med 1978;88:468-73 23. Martinez AI, Garcia CA, Halks-Miller MH, Arce-Vela R. Granulomatous amebic encephalitis presenting as a cerebral mass lesion. Acta Neuropathol (Berl) 1980;51:85-91 24. Hoffmann EO, Garcia C, Lunseth J, McGarry P, Coover 1. A case of primary amebic meningoencephalitis. Light and electron microscopy, and immunohistologic studies. Am I Trop Med Hyg 1978;27:29-38
RID 1991;13 (Suppl 5)
Infection of the Central Nervous System Due to Acanthamoeba A. Julio Martinez
From the Pathology Department (Neuropathology), PresbyterianUniversity Hospital, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
It is wellestablished that Acanthamoeba castellanii, Acanthamoeba culbertsoni, Acanthamoeba polyphaga, and probably other species of free-living ~meb~ ar~ virul~nt op~ortunists ~pable
For many years Entamoeba histolytica was thought to be the only ameba pathogenic for mammals. In the last few years, however, it has become apparent that several species of freeliving amebas can infect human beings and animals and that death may result from such infections [1]. Two main genera of free-living amebas have been found to infect humans: Naegleria and Acanthamoeba. Naegleria fowleri may produce an acutely fatal infection of the CNS that is known as primary amebic meningoencephalitis (PAM) and resembles fulminant bacterial meningitis. PAM usually occurs in healthy young individuals with a history of either swimming in or recent nasal contact with contaminated water [2~. Species of Acanthamoeba may cause a subacute or chrome CNS infection known as granulomatous amebic encephalitis (GAE) [3]. In addition, acanthamoebas may produce acanthamoebic keratitis [4], pneumonitis, and subacute granulomatous dermatitis. CNS infection due to Acanthamoeba species usually occurs in patients who have impaired defense mechanisms or who are ill and debilitated and, in many cases, are being treated with immunosuppressive drugs or broad-spectrum antibiotics. The infections produced by Acanthamoeba species are considered to be opportunistic [3]. Considerable progress has been made in the recognition of the symptoms and the histomorphologic features of PAM and GAE, and more cases are likely to be reported in the future as the medical community becomes more aware of procedures for isolating, cultivating, and identifying the causative organisms.
Reprints and correspondence: Dr. A. 1. Martinez, Division ofN.europathoiogy, Presbyterian-University Hospital, Pittsburgh, Penns~lvama 15213. This work was supported in part by the Pathology Education and Research Foundation of the Department of Pathology, University of Pittsburgh. Reviews of Infectious Diseases 1991;13(8uppl5):8399-402 © 1991 by The University of Chicago. All rights reserved. 0162-0886/91/1302-0070$02.00
Epidemiologic Aspects of GAE The epidemiology of Acanthamoeba species is both complex and intriguing. Acanthamoebas are inhabitants of soil, water, sewage and wastes, thermally polluted waters, cooling towers, air-conditioning filters, and fecal material from birds, reptiles, and mammals [4-6]. The air and dust may carry amebic cysts. Infections have been observed in humans, cattle, reptiles, dogs, and fish. The increased interest in these amebas bas led to descriptions of new species isolated from aquariums, swimming pools, throat swabs, human stools, contaminated cell cultures, marine sediments, and contact lenses [7-13]. Acanthamoeba castellanii, Acanthamoeba culbertsoni, Acanthamoeba polyphaga, and Acanthamoeba palestinensis are the better-known species. Some newly identified species include Acanthamoeba royreba, Acanthamoeba lenticulata, Acanthamoeba hatchetti, Acanthamoeba griffini, and Acanthamoeba triangularis, but there are still several unidentified species that probably are responsible for infections of the CNS, cornea, respiratory tract, and/or skin [14, 15]. Experimental studies in several laboratories have shown that some acanthamoebas may ingest the bacteria responsible for Legionnaire's disease and act as aerosol carriers [6]. Other studies of factory workers who have become ill with "humidifier" fever or with respiratory symptoms such as hypersensitivity pneumonitis have shown that acanthamoebas can be present in water from cooling towers and in air filters [13]. So far, no animal reservoir of free-living amebas has been demonstrate as a carrier.
eNS Involvement by Acanthamoeba Species The route of invasion and penetration of acanthamoebas into the eNS in cases of GAE appears to be hematogenous, probably with a primary focus in the lower respiratory tract or
Downloaded from http://cid.oxfordjournals.org/ at UQ Library on August 15, 2015
of producing disease in humans and animals. Human infections involving braIn: eyes,. skin, and lungs have been reported from all continents. ~entr~l nerv~~s sys~m ~~NS) infection due to Acanthamoeba species usually occurs in chromcally ill, debilitated individuals, some of them receiving immunosuppressive therapy or taking broad-spectrum antibiotics. T~e disease ~?S a protracted, insidious clinical course and is known as granulo~atous ameb~c encephaliti». Histopathologically, Acanthamoeba species may produce a m~tifocal, c~omc, or subacute granulomatous encephalitis, with trophozoites and cysts present In C~S les~ons. The portal of entry of the amebas into the CNS is probably the respiratory tract or a skin lesion, and the organisms reach the CNS by hematogenous spread. As of 1 January 1989,about SO cases of granulomatous amebic encephalitis had been reported worldwide, 27 in the United States alone.
S400
Martinez
through an ulceration of the skin. In experimental animals the olfactory neuroepithelium has also been found to be a possible portal of entry [2]. The incubation period of GAE is unknown but is probably longer than 10 days [2]. The ability of acanthamoebas to produce necrosis of brain tissue is probably due to an enzymatic action induced by lysosomal hydrolases and a phospholipase that can degrade phospholipids of the myelin sheaths [14].
Clinical Manifestations of GAE
Pathologic Features of GAE Macroscopic appearance ofinfected CNS tissues. In GAE the cerebral hemispheres show moderate edema. The leptomeninges may contain a moderate amount of purulent exudate, with minimal cloudiness over the most affected cortical areas. In the less affected regions, the leptomeninges are transparent [2]. There are foci of encephalomalacia and softening associated with discrete or confluent areas of hemorrhagic necrosis in occipital, parietal, temporal, or-less often-frontal lobes. The lesions are usually multifocal, located in the posterior fossa structures, midbrain, thalamus, brainstem, corpus callosum, and cerebellum. The cervical portion of the spinal cord is occasionally affected. Bilateral uncal notching and
cerebellar tonsillar herniations may be prominent features. The olfactory bulbs and base of the frontal lobes are usually spared [2]. Microscopic findings in infected CNS tissues. The lesions of GAE due to Acanthamoeba species in the CNS may vary from case to case in location, extent, composition of cellular elements, and density of trophozoites and cysts. As a rule, the lesions are most numerous in the basal ganglia, midbrain, brainstem, and cerebral hemispheres [2]. Usually there is a focal, rather modest, chronic or subacute leptomeningitis, most of which is close to parenchymal lesions. The characteristic lesion in the CNS parenchyma is a necrotizing subacute or chronic granulomatous encephalitis in which trophozoites and cysts may be found in association with "giant cells;' some of them engulfing amebas. The amebic trophozoites and cystsmay be present in large numbers, mainly in perivascular spaces of the affected areas. Sometimes clusters of trophozoites are seen without an accompanying inflammatory response. Gliosis around necrotic areas is usually modest. Phagocytic nodules may be observed. Foci of recent hemorrhages may be seen, usually within the necrotic areas. In some cases there is severe angiitis (panarteritis), with perivascular cuffing by lymphocytes, some plasma cells, few macrophages, and rare eosinophils.Fibrinoid necrosis and thrombosis constitute an interesting feature in some instances. Amebic trophozoites and cysts may be found piercing the vascular wall. In some cases, particularly in patients with AIDS, the absence of a granulomatous reaction indicates severely compromised host defense mechanisms. At postmortem, involvement of viscera other than the CNS may be found, suggestinga premortem hematogenous dissemination of trophozoites and cysts. The organs involved may be subcutaneous tissue and skin, liver, lungs, kidneys, adrenals, pancreas, prostate, lymph nodes, and myometrium [2, 33].
Diagnosis of GAE Laboratory methods, differential diagnosis, direct microscopic diagnosis, and cultures. The CSF findings may be of value in the diagnosis of acanthamoeba infection [34]. Pleocytosis is the rule, with lymphocytic predominance. The opening pressure is slightly elevated; the glucose concentration is usually low and the protein concentration moderately high. Polymorphonuclear leukocyte counts are also elevated but not as high as in PAM due to N. fowleri. The CSF is not as frankly purulent as in bacterial meningitis. Amebic trophozoites may be present in the CSF [32]. An important laboratory procedure for the diagnosis of infections due to free-living amebas is the microscopic examination of CSF. Trophozoites may be found in cases of PAM due to N. fowleri. Motile amebas are sought by placing one or two drops of unstained CSF (which has been centrifuged at low speed) on a glass slide, coverslip, or hanging drop and then using an ordinary light microscope with lowered diaphragm for phase-contrast or dark-field illumination. Amebas
Downloaded from http://cid.oxfordjournals.org/ at UQ Library on August 15, 2015
Symptoms and signs. Clinical symptoms of GAE due to Acanthamoeba species are mainly those of focal or localized encephalopathy with subsequent severe meningeal irritation and encephalitis, i.e., mental status abnormalities and behavioral changes (confusion, irritability, somnolence, hallucinations, and dizziness), seizures, headache, hemiparesis, aphasia, cranial nerve palsies, fever, stiff neck, visual disturbances (diplopia and blurred vision), anorexia, nausea and vomiting, ataxia, papilledema, coma, and death [16-40]. Clinical course: morbidity, mortality, and prognosis. The clinical presentation of GAE is insidious and may mimic that of bacterial leptomeningitis, tuberculous meningitis, or viral encephalitis. Some patients may present initially with respiratory infection or with gastrointestinal symptoms; some with a chronic ulceration of the skin or with skin nodules; and others with symptoms mimicking a space-occupying mass, a brain abscess, or a brain tumor. In the latter cases craniotomies for a brain biopsy and frozen section may unravel the diagnosis. Clinical setting, risk, and predisposing factors. GAE due to Acanthamoeba species may occur in individuals who are pregnant, chronically ill and debilitated, chronically alcoholic, or malnourished. Some patients have underlying diseases such as skin ulcers or skin trauma, lymphomas, diabetes mellitus, cirrhosis of the liver or other hepatic disease, AIDS, gammaglobulinemia, and glucose-6-phosphate dehydrogenase deficiency [2]. Patients at risk are those receiving treatment with steroids, antibiotics, and immunosuppressive drugs (e.g., agents used to prevent organ rejection in transplant recipients).
RID 1991;13 (Suppl 5)
RID 1991;13 (Suppl 5)
CNS Acanthamoeba Infection
differentiation of trophozoites of N. fowleri and Acanthamoeba species in CNS tissue may be difficult by light microscopy, but trophozoites of Acanthamoeba are larger (l5-4~ I'm in diameter). They characteristically contain a centrally placed, dense karyosome or nucleolus that is surrounded by a clear nuclear halo of chromatin and a double nuclear membrane. The nucleus measures "-15-10 I'm in diameter. The cytoplasm is abundant with slender hyaline projections or acanthopodia. Clear and dense vacuoles and dense granules are seen in the cytoplasm. The mean diameter of cystsof Acanthamoeba species is "-115-20 I'm. Diagnosis by electron microscopic techniques. The ultrastructural features of amebic trophozoites and cysts of Acanthamoeba species have been described for postmortem material as well as for axenic cultures and in the experimental disease in animals. Electron microscopic examination of pathogenic acanthamoebas may reveal numerous oval, round, and "dumbbell-shaped" mitochondria. Rough and smooth endoplasmic reticulum is abundant. The Golgi apparatus is prominent. Food vacuoles containing myelin figures are conspicuous. Some empty vacuoles are also seen. The karyosome is composed of dense RNA, and the nuclear chromatin is finely granular and surrounded by a double membrane. During nuclear division the karyosome may divide in two and the nuclear membrane is absent - primarily during the late prophase. The cysts of Acanthamoeba species have a stellate, spherical, or polyhedral shape, with double wrinkled walls or a solid, compact wall. Ostioles and opercula in which excystation takes place may be seen. The cyst walls are laminated and united at the level of opercula.
Treatment of GAE At this time no effectivetreatment is available for CNS acanthamoeba infection. In contrast, ophthalmic infections such as acanthamoeba keratitis have been treated with success in some cases. For example, propamidine isethionate (Brolene), an ophthalmic medication that can be obtained over the counter in England, has been reported to be effective in the treatment of acanthamoeba keratitis. Neomycin also appears to be effective in combination with propamidine isethionate. The efficacy of these drugs in GAE is not known. Sulfadiazine is probably ineffective, perhaps because of the inadequacy of the host's immune system. Ketoconazole appears to be effective both in vitro and in vivo (in experimental animal models) for treatment of CNS infections. Acridine derivatives and paromomycin seem promising in vitro, but the use of these compounds is still in the experimental stage [43, 44].
References 1. Martinez AJ. Free-living amebas: natural history, prevention, pathology, and treatment of disease. Boca Raton, FL: CRC Press, 1985:156 2. Martinez AJ. Free-living amoebas: pathogenic aspects. A review. Protozoology Abstracts 1983;7:293-306
Downloaded from http://cid.oxfordjournals.org/ at UQ Library on August 15, 2015
are refractile and can be seen moving. They must be differentiated from lymphocytes, monocytes, polymorphonuclear leukocytes, and macrophages. Naegleria species are highly motile, with lobopodia erupting rapidly. Motility is one of the most important features to observe. Acanthamoeba species are sluggish and may be confused with macrophages. A few cases of acanthamoeba infection have been diagnosed by microscopic observation of the motile but sluggish organisms [34]. Giemsa- or Wright-stained smears of centrifuged CSF offer the advantage of showing the characteristic morphology of the amebas that differentiates them from lymphocytes, erythrocytes, and polymorphonuclear leukocytes. An abundance of erythrocytes may suggest PAM due to N. fowleri, which is characterized by necrosis and hemorrhage [1]. Cultures can be prepared from the CSF, or from brain tissue, but few cases of GAE have been diagnosed by means of such cultures [34]. Acanthamoeba species are considered synonymous with Hartrnannella by some protozoologists. This group of organisms is characterized by a centrally placed, dense nucleolus that is surrounded by a clear nuclear halo and abundant cytoplasm. Usually gram-stained smears and bacterial cultures are negative. Amebas can easily be seen in tissues stained with hematoxylin and eosin. Cysts are better seen with periodic acid-Schiff hematoxylin or Grocott methenamine silver staining. Retrospective diagnosis of GAE. The great majority of cases of GAE have been diagnosed postmortem by the detection of amebic trophozoites and cysts in sections of CNS tissue embedded in paraffin and stained with hematoxylin and eosin [41]. Immunofluorescent antibody and immunoperoxidase methods. When no cultures have been prepared or isolates obtained, the indirect immunofluorescent antibody test (IFAT) or immunoperoxidase staining (IPAS) of CNS tissue fixed in formalin and embedded in paraffin is valuable for the identification and localization of trophozoites and cysts [42]. The tissues are treated with specific, labeled antiserum. However, there is considerable antigenic overlap between species of the genus Acanthamoeba (A. castellanii, A. culbertsoni, A. polyphaga, and Acanthamoeba astronyxis) and other free-living amebas. The use of IFAT and IPAS may permit more precise identification than is possible by morphologic study of the trophozoites and cysts. In addition, immunodiffusion or immunoelectrophoretic analysis of water-soluble proteins of the amebic trophozoites from cultures may be used for the identification and comparison of free-living strains of ameba. In some cases the IPAS or IFAT is inconclusive and the diagnosis can be reasonably established only by observation of the morphologic features of the trophozoites and cysts and the histopathologic features of the affected CNS tissue. The diagnosis of possible vahlkampfia infection may be suggested on the basis ofbinucleation of some of the trophozoites in the CNS lesions or of negative IFAT and IPAS results in tests for several Acanthamoeba species and N. fowleri. Diagnosis by light microscopic techniques. Morphologic
5401
5402
Martinez
25. Cox EC. Amebic meningoencephalitis caused by Acanthamoeba species in a four month old child. J S C Med Assoc 1980;76:459-62 26. Wessel HB, Hubbard I, Martinez AI, Willaert E. Granulomatous amebic encephalitis (GAE) with prolonged clinical course: CT scan findings, diagnosis by brain biopsy and effect oftreatment [abstract]. Neurology (Minneap) 1980;30:442 27. Gullett I, Mills I, Hadley K, Podemski B, Pitts L, Gelber R. Disseminated granulomatous Acanthamoeba infection presenting as an unusual skin lesion. Am J Med 1979;67:891-6 28. Martinez AI, Sotelo-AvilaC, Alcala H, Willaert E. Granulomatous encephalitis, intracranial arteritis, and mycotic aneurysm due to a freeliving ameba. Acta Neuropathol (Berl) 1980;49:7-12 29. Grunnet ML, Cannon GH, Kushner JP. Fulminant amebic meningoencephalitis due to Acanthamoeba. Neurology (Minneap) 1981;31:174-7 30. Carter RF, Cullity GJ, Ojeda VJ, Silberstein P, Willaert E. A fatal case of meningoencephalitis due to a free-living amoeba of uncertain identity-probably Acanthamoeba sp. Pathology 1981;13:51-68 31. Borochovitz D, Martinez AI, Patterson GT. Osteomyelitis ofa bone graft of the mandible with Acanthamoeba castellaniiinfection. Hum Pathol 1981;12:573-6 32. Cleland PG, Lawande RV, OnyemelukweG, Whittle He. Chronic amebic meningoencephalitis. Arch Neurol 1982;39:56-7 33. Martinez AI. Acanthamoebiasis and immunosuppression. Case report. I Neuropathol Exp Neurol 1982;41:548-57 34. Lalitha MK, Anandi V, Srivastava A, Thomas K, Cherian AM,. Chandi SM. Isolation of Acanthamoeba culbertsoni from a patient with meningitis. I Clin Microbiol 1985;21:666-7 35. Gonzalez MM, Gould E, Dickinson G, Martinez AI, Visvesvara G, Cleary TI, Hensley GT. Acquired immunodeficiency syndrome associated with Acanthamoeba infectionand other opportunistic organisms. Arch Pathol Lab Med 1986;110:749-51 36. Wiley CA, Safrin RE, Davis CE, Lampert PW, Braude AI, Martinez AI, Visvesvara GS. Acanthamoeba meningoencephalitis in a patient with AIDS. I Infect Dis 1987;155:130-3 37. Ofori-Kwakye SK, Sidebottom DG, Herbert J, Fischer EG, Visvesvara GS. Granulomatous brain tumor caused by Acanthamoeba. Case report. I Neurosurg 1986;64:505-9 38. Rutherfoord GS. Amoebic meningo-encephalitis due to a free-living amoeba. S Afr Med J 1986;69:52-5 39. Matson DO, Rouah E, Lee Rf, Armstrong D, Parke IT, Baker C1. Acanthameba meningoencephalitis masquerading as neurocysticercosis. Pediatr Infect Dis J 1988;7:121-4 40. Harwood CR, Rich GE, McAleer R, Cherian G. Isolation of Acanthamoeba from a cerebral abscess. Med I Aust 1988;148:47-9 41. Willaert E, Stevens AR, Healy GR. Retrospective identification of Acanthamoeba culbertsoni in a case of amoebic meningoencephalitis. J Clin Pathol 1978;31:717-20 42. Willaert E, Stevens AR. Indirect immunofluorescent identification of Acanthamoeba causing meningoencephalitis. Pathol BioI 1976;24: 545-7 43. Steven AR, Willaert E. Drug sensitivity and resistance of four Acanthamoeba species. Trans R Soc Trop Med Hyg 1980;74:806-8 44. Osuna A, Rodriguez-Santiago11,Ruiz-Perez L-M, Gamarro F, Castanys S, Giovannangeli G, Galy AM, Galy IP, Soyfer JC, Barbe 1. Antiamebic activity of new acridinic derivatives against Naegleria and Acanthamoeba species in vitro. Chemotherapy 1987;33:18-21
Downloaded from http://cid.oxfordjournals.org/ at UQ Library on August 15, 2015
3. Martinez AI. Is acanthamoebic encephalitis an opportunistic infection? Neurology (New York) 1980;30:567-74 4. Lewis EJ, Sawyer TK. Acanthamoeba tubiashi N. Sp., a new species of fresh-water Amoebida (Acanthamoebidae). Transactions of the American Microscopy Society 1979;98:543-8 5. SawyerTK, Visvesvara GS, Harke EA. Pathogenicamoebas from brackish and ocean sediments, with a description of Acanthamoeba hatchetti, n. sp. Science 1977;196:1324-5 6. RowbothamTI, et al. Preliminary report on the pathogenicityof Legionella pneumophila for freshwater and soil amoebae. J Clin Pathol 1980;33:1179-83 7. Casemore DP. Contamination of virological tissue cultures with a species of free-living soil amoeba. J Clin Pathol 1969;22:254-7 8. Willaert E, Stevens AR, Tyndall RL. Identification of Acanthamoeba culbertsoni from cultured tumor cells. Protistologica 1978;14:319-36 9. Wang SS, Feldman HA. Isolation of Hartmannella species from human throats. N Engl J Med 1967;277:1174-9 10. Visvesvara GS, Mirra SS, Brandt FH, Moss DM, Mathews HM, Martinez AI. Isolation of two strains of Acanthamoeba castellanii from human tissue and their pathogenicity and isoenzyme profiles. J Clin Microbiol 1983;18:1405-12 11. Tyndall RL, Willaert E, Stevens A, Nicholson A. Pathogenic and enzymatic characteristics of Acanthamoeba isolated from cultured tumor cells. Protistologica 1979;15:17-22 12. DeJonckheere JE Pathogenic and nonpathogenic Acanthamoeba spp. in thermally polluted discharges and surface waters. J Protozool 1981;28:56-9 13. Edwards JH, Griffiths AI, Mullins J. Protozoa as sources of antigen in "humidifier fever." Nature 1976;264:438-9 14. Costas M, Griffiths AF. Enzyme composition and the taxonomy of Acanthamoeba. J Protozool 1985;32:604-7 15. Page Fe. Re-definition of the genus Acanthamoeba with descriptions of three species. I Protozool 1967;14:709-24 16. Kernohan J, Magath TB, Schloss GT. Granuloma of brain probably due to Endolimax williansi (Iodamoeba butschliiy. Archives of Pathology 1960;70:576-80 17. Robert VB, Rorke LB. Primary amebic encephalitis, probably from Acanthamoeba. Ann Intern Med 1973;79:174-9 18. Jager BV, Stamm WP. Brain abscesses caused by a free-living amoeba probably of the genus Hartmannella in a patient with Hodgkin's disease. Lancet 1972;2:1343-5 19. Bhagwandeen SB, Carter RF, Naik KG, Levitt D. A case of Hartmannellid amebic meningoencephalitis in Zambia. Am J Clin Pathol 1975;63:483-93 20. Ringsted I, Jager BV, Suk D, Visvesvara GS. Probable Acanthamoeba meningoencephalitis in a Korean child. Am J Clin Pathol 1976; 66:723-30 21. Martinez AI, Sotelo-Avila C, Garcia-Tamayo J, 'Iakano-Moron I, Willaert E, Stamm WP. Meningoencephalitis due to Acanthamoeba SP: pathogenesis and clinico-pathological study. Acta Neuropathol (Berl) 1977;37:183-91 22. Duma RJ, Helwig WB, Martinez AI. Meningoencephalitis and brain abscess due to free-living amoebae. Ann Intern Med 1978;88:468-73 23. Martinez AI, Garcia CA, Halks-Miller MH, Arce-Vela R. Granulomatous amebic encephalitis presenting as a cerebral mass lesion. Acta Neuropathol (Berl) 1980;51:85-91 24. Hoffmann EO, Garcia C, Lunseth J, McGarry P, Coover 1. A case of primary amebic meningoencephalitis. Light and electron microscopy, and immunohistologic studies. Am I Trop Med Hyg 1978;27:29-38
RID 1991;13 (Suppl 5)
