1985-Acanthamoeba, an opportunistic microorganism A review

Review,

A. J. M a r t i n e z , K. J a n i t s c h k e

Acanthamoeba, an Opportunistic Microorganism: A Review

Summary: Granulomatous amebic encephalitis due to Acanthamoeba spp. usually occurs in chronically ill and debilitated individuals. Some of these patients may have received immunosuppressive therapy. An- other infection due to Acanthamoeba spp. has been corneal ulcerations which usually occur after minimal trauma to the corneal epithelium (1). In contrast, primary amebic meningoencephalitis due to Naegleria fowleri usually occurs in healthy, young individuals with a history of swimming in heated swimming pools, in manmade lakes or with recent contact with contami- nated water and practising water-related sports. Sub- clinical infections due to flee-living amebas are probably common in healthy individuals with the protozoa living as "normal flora" in the nose and throat. It is possible that in humans, antibodies and cell-mediated immunity protect the host in such ordinary circum-

Zusammenfassung: Acanthamoeba, ein opportunisti- scher Mikroorganismus. Die granulomat6se Am6ben- encephalitis durch Acanthamoeba spp. tritt in der Re- gel bei chronisch kranken und geschw/ichten Personen auf. Bei manchen dieser Patienten kann eine immun- suppressive Therapie vorausgegangen sein. Eine andere Infektion durch Acanthamoeba spp. tritt in Form von Hornhautulzera, gew6hnlich nach geringfiigiger Verletzung des Hornhautepithels auf (1). Dagegen ist die prim/ire Am6ben-Meningoencephalitis durch Nae- gleria fowleri eine Krankheit junger, gesunder Perso- nen, die in geheizten Schwimmb/idern oder ktinst- lichen Seen geschwommen sind, ktirzlich mit kontami- niertem Wasser in Beriihrung gekommen sind oder Wassersport treiben. Subklinische Infektionen durch freilebende Am6ben sind bei gesunden Personen wahrscheinlich h/iufig, dabei leben die Protozoen als ,,normale Flora" in der Nase und im Rachen. Es ist m6glich, dab unter natiirlichen Umst/inden beim Men- schen ein Schutz gegen invasive Infektionen durch An-

stances against invasive infection. In debilitated and chronically ill individuals, depressed cellmediated immunity may allow these protozoa to proliferate, allowing a fulminant "opportunistic" infection to develop. In the case of acanthamoebic keratitis, it is important to keep in mind that the temperature and moist envi- ronment of the eye serve as a good medium for the growth and proliferation of the amebas and is not necessarily associated with immunosuppression but rather with trauma. This review confirms that opportunistic free-living amebic infections occur with in- creased frequency in patients treated with steroids, ra- diotherapy, chemotherapeutic drugs or with broad- spectrum antibiotics and suggest that the mechanism of such infection may be depressed cell-mediated immunity or some other alteration of the immune system, like acquired immunodeficiency syndrome (AIDS).

tik6rper und zellvermittelte Immunit/it besteht. Bei geschw/ichten und chronisch kranken Personen k6n- nen die Protozoen infolge der Abwehrschw/iche proli- ferieren und eine fulminante ,,opportunistische" In- fektion verursachen. Bei der Acanthamoeba-Keratitis ist zu bedenken, dab die Temperatur und das feuchte Milieu des Auges ftir das Wachstum und die Prolifera- tion der Am6ben ein gutes Medium darstellen, und diese Erkrankung nicht notwendigerweise mit Immun- suppression sondern eher mit Trauma assoziiert ist. Die vorliegende 12Ibersicht best/itigt, dab opportunistische Infektionen durch freilebende Am6ben zuneh- mend h~iufiger bei Patienten, die mit Steroiden, Be- strahlung, Chemotherapeutika oder Breitspektrum- antibiotika behandelt werden, auftritt; es ist anzuneh- men, dab fiir die Pathogenese dieser Erkrankung eine Beeintr~ichtigung der zellvermittelten Immunit/it oder andere Anderungen des Immunsystems wie das erwor- bene Immundefektsyndrom (AIDS) yon Bedeutung sind.

Introduction

Infectious diseases continue to be the leading cause of morbidity and mortality in developing and industrial so- cieties, particularly in individuals with compromised host defenses. Such "compromised hosts" are subject to a wide variety of "opportunistic" infections (2, 3). Most of these infections are due to bacteria, fungi or viruses and least often due to protozoa and helminths. To this list must now be added a subacute or chronic granulomatous encephalitis, keratitis, dermatitis, brain abscesses and

pneumonitis due to Acanthamoeba spp. (Acanthamoeba castellanii, Acanthamoeba culbertsoni, Acanthamoeba polyphaga, Acanthamoeba astronyxis [in some publications considered as belonging to the genus Hartmannella and

Received: 20 May 1985/Accepted: 30 August 1985

A. J. Martinez, M.D., Pathology Department, Presbyterian-University Hospital, University of Pittsburgh, Pittsburgh, Penn. 15213, USA; Prof. Dr. K. Janitschke, Robert Koch Institute, Department of Clinical Parasitology, Nordufer 20, D-1000 Berlin. Reprint requests: Prof. Dr. K. Janitschke.

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A. J. Martinez, K. Janitschke: Acanthamoeba, an Opportunistic Microorganism

named as Hartmannella culbertsoni, Hartmannella castellanii and Hartmannella polyphaga]), a type of free living or amphizoic ameba. Patients with acquired immunodeficiency syndrome (AIDS) may also be included in the list ,of susceptible individuals to develop Acanthamoeba spp. infections. The objective of the present review is to sur- vey the current status of Acanthamoeba spp. infection and to assess the clinico-neuropathological characteristics of human infections and to obtain a better understanding of the pathogenesis, clinical mode of presentation, diagnostic methods, epidemiology, and neuropathological features of the disease.

Clinical Aspects: Signs and Symptoms

The patients susceptible to granulomatous amebic encephalitis (GAE) range in age from children to the elderly. Apparently, there is no sex preference for the amebic disease to appear. The clinical course or duration of the ill- ness ranges from one week to about 120 days (2). Clinical symptoms of GAE due to Acanthamoeba spp. are mainly those associated with focal or localized encephalopathy with subsequent severe meningeal irritation and encephalitis: mental status abnormalities and behavioral changes, like confusion, irritability, somnolence, hallucinations and dizziness; seizures, headache, hemiparesis, aphasia and cranial nerve palsies, fever, stiff neck; visual disturb- ances like diplopia and blurred vision; anorexia, nausea and vomiting, ataxia, and papilledema, progressing to co- ma and death. The clinical presentation may mimick bacterial leptomeningitis, tuberculous or cryptococcal meningitis, or viral encephalitis. Some patients may present initially with respiratory infection or with gastrointestinal symptoms. Other patients may present initially with a chronic ulceration of the skin or with another type of skin lesion. Some patients may disclose symptoms mimicking a "space occupying mass" or "brain abscess". In contrast, cases of primary amebic meningoencephalitis (PAM) due to Naegleriafowleri are those associated with a severe dif- fuse meningeal irritation, widespread encephalitis, usually without localizing symptoms or signs (4). Typically, GAE due to Acanthamoeba spp. occurs in debilitated individuals with some associated underlying diseases such as skin ulcers of skin trauma, diabetes mellitus, cirrhosis of the liver or some other liver disease, in patients undergoing immunosuppressive therapy for hema- tologic malignancies such as malignant lymphoma, Hodg- kin's disease, gamma globulinemia, G6PD deficiency, pharyngitis, otitis and pneumonitis or other pulmonary disease, chronic renal failure, and collagen-connective tissue disease (4) (Table 1). Predisposing factors for the establishment of acanthamoebic infection are included in Table 2. There is no history of water-related sport activities in any patient with granulomatous amebic encephalitis, but in some cases of acanthamoebic keratitis there has been a history of water contact with the eye (1). The CSF findings may be of value in suggesting the diag-

Table 1 : Associated disorders in 33 patients with granulomatous amebic encephalit is (GAE) (2).

Skin ulcers Cirrhosis of liver and hepatitis Abnormal gammaglobulin Cerebral infarct Pneumonitis Diabetes mellitus Hodgkin's disease or lymphoma G6PD deficiency Chronic renal failure Otitis media and pharyngitis Unknown

7 4 1 1 3 1 2 1 2 1 8

21 12 3 3 9 3 6 3 6 3

24

nosis of Acanthamoeba infection. Pleocytosis is the rule with lymphocytic predominance. The glucose is usually low and proteins very high. Polymorphonuclear leukocytes are also elevated but not as high as in PAM due to N. fowleri and not as frankly purulent as in bacterial meningitis.

Neuropathological Aspects

The leptomeninges may contain a moderate amount of purulent exudate with minimal cloudiness mainly over the most affected cortical areas. In the less affected regions, the leptomeninges are transparent. When the ameba reaches the CNS, there is virtually unimpeded spread of the organisms within the affected areas but in a centrifu- gal manner indicating a hematogenous route to reach the CNS (5, 6). The cerebral hemispheres show moderate edema. There are foci of encephalomalacia associated with areas of hemorrhages. The lesions are usually multifocally located in the midbrain, thalamus, brainstem, corpus callosum, or cerebellum. Discrete or confluent areas of encephalomalacia may be present (Figure 1). The upper portion of the spinal cord is occasionally affected. Sometimes the lesions may present as a space occupying mass or as a brain tumor. This is a very important clinical presentation, which may mimick a brain tumor or an abscess. Bilateral uncal notching and cerebellar tonsillar herniations may be prominent features. The olfactory bulbs and base of frontal lobes are usually spared.

Table 2: Predisopsing factors in 33 patients with granulomatous amebic encephalit is (GAE) (11).

Antibiotics Steroids Chemotherapy Radiation therapy Alcoholism Pregnancy Peritoneal dialysis Splenectomy Renal transplant

12 36 15 45 8 24 3 9 4 12 2 6 2 6 1 3 1 3

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' ~ "~ ~ ~ - " ' • "~e, ~ . .

Figure 1 : Cross sections of cerebellum and brainstem with large areas of hemorrhagic, necrotizing encephalomalacia. A 38-year-old white man, receiving immunosuppressive therapy for a renal transplant.

Histopathology of the CNS Lesions

Although the lesions of GAE due to Acanthamoeba spp. in the CNS are of the same character in all the cases, they may vary moderately from case to case in location, ex- tent, composition of cellular elements, trophozoites, and cyst density. As a rule, the lesions are most evident in the cerebellum, midbrain, and brainstem; often the basal ganglia may be severely affected, and occasionally the an- terior portion of the cerebral hemispheres. Usually there is a focal, rather modest chronic or subacute leptomeningitis, mainly near or very close to the location of the main lesions. The characteristic lesion in cerebral hemispheres, brainstem, and cerebellum is a necrotizing granulomatous subacute or chronic encephalitis in which trophozoites and cysts may be found, associated with "giant cells" some of them engulfing amebas. The amebic trophozoites and cysts may be present in large numbers in perivascular spaces of the affected areas (Figure 2). Sometimes clusters of trophozoites may be seen without inflammatory response. Gliosis is usually modest. Sometimes "microglial" nodules may be observed. Foci of recent hemorrhages may be seen where the most necrotic areas are found. In most cases, there is a severe angiitis (panarteritis) with perivascular cuffing by lymphocytes, some plasma cells, few macrophages and a variable number of eosinophils. Fibrinoid necrosis and thrombosis may be a prominent feature. Amebic trophozoites may be present throughout the vascular wall and occasional giant cells and/or trophozoites may be present within the lumens and may or may not be thrombosed. The localization and distribution of the lesions suggest that the mechanism by which the protozoa reaches the CNS is via the blood stream from a primary focus, probably skin, or lower respiratory tract (Figure 3). The free- living amebas (Acanthamoeba spp.) may produce "ame-

Figure 2: Amebic trophozoites and cysts (Acanthamoeba castellanii) infiltrating the arterial wall. The blood vessels are surrounded by edematous and necrotic CNS tissue (H & E X 125).

bic dermatitis and cellulitis", "amebic pneumonitis" and acanthamoebic keratitis. At post mortem, in some cases, visceral involvement other than the CNS may be found. This probably suggests a premortem hematogenous dissemination of trophozoites and/or cysts. The organs involved may be lungs (probably the primary lesion or portal of entry), liver, skin, kidneys, adrenals, pancreas, prostate, lymph nodes, and myome- trium. PAM due to N. fowleri by contrast shows severe acute necrotizing and hemorrhagic meningoencephalitis mainly

Figure 3: Pulmonary lesion containing numerous hyphae of Candida albicans. Amebic trophozoites and cyst can be seen between the fungi. Same case as Figures 1 and 2 (me- thenamine silver X 250).

involving the base of the frontal lobes and posterior fossa structures. The lesions are usually located on the surface of the cerebral hemispheres, cerebellum, and brainstem. Occasionally, the ventricular system is affected, showing an acute choroid plexitis and ependymitis. The portal of

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entry of N. fowleri to the CNS is through the cribriform plate by phagocytosis of the ameba by the substentacular cells of the olfactory neuroepithelium.

Morphological Characteristics of Acanthamoeba spp,

By light microscopy, trophozoites of N. fowleri and Acanthamoeba spp. in CNS tissue may be morphological- ly indistinguishable; however, trophozoites of Acanth- amoeba are larger. The dimensions of trophozoites of Acanthamoeba spp. vary from 15 to 45 Ixm in diameter (Figure 4). They contain a characteristically centrally placed, dense karyosome (nucleolus) surrounded by a clear nuclear halo and a thin nuclear membrane measur- ing about 5 to 10 ~m in diameter. The cytoplasm is abundant with numerous clear and dense vacuoles and dense granules with some slender hyaline projections or acanth- opodia. During nuclear division, the karyosome may be divided in two and the nuclear membrane is absent, particularly during late prophase. The Acanthamoeba spp. cysts reveal a stellate, spherical or polyhedral shape with double and/or wrinkled wall. They usually measure about 15 to 20 ~tm in average diameter. Ostioles and opercula may be seen in which excystation takes place (7-9).

e4 ;

Figure 4: lrophozoites of Acanthamoeba caste/laniLshow- ing the characteristic dense nucleolus surrounded by a clear nuclear halo, and abundant granular cytoplasm. Axe- nic culture. Plastic embedded (I ~rn section, toluidine blue x 550).

The ultrastructural features of amebic trophozoites and cysts of pathogenic A. culbertsoni and A. castellanii may reveal numerous oval, round and "dumbbell-shaped" mi- tochondria. The rough and smooth endoplasmic reticulum is abundant. The Golgi apparatus is prominent. Food vacuoles containing "myelin figures" are conspicuous. Some emp- ty vacuoles are also seen. The karyosome is composed of dense RNA and the nucleus is finely granular and surrounded by a double membrane. The cyst walls are lami- nated and united at the level of the opercula. They con-

tain a densely packed electron-dense and granular mate- rial. When no culture or isolation has been made, the indirect immunofluorescent or immunoperoxidase staining of CNS tissue fixed in formalin and embedded in paraffin is a valuable tool in the identification and localization of trophozoites.

Comment

The increasing frequency with which protozoal amebic infections are being recognized in all parts of the world re- flects many factors: wider awareness of their occurrence, better diagnostic facilities, more accurate diagnosis, the increasing prevalence of "opportunistic" infections due to facultatively pathogenic organisms. It seems likely that Acanthamoeba may exist and are present in humans as part of the "normal flora" or in the en- vironment (10-13). A large number of amebas have been isolated in culture from samples of human origin. With culture procedures, it is difficult to distinguish between a sample with active trophozoites and growth resulting from the presence of a random cyst. Even if trophozoites are seen, it may not be possible to distinguish pathogens from opportunistic residents in sites like the nasopharynx or digestive tract. By using tissue cultures similar to those used to isolate viruses, a number of strains of two species of Acanthamoeba and one of Hartmannella have been isolated from human throats. An ameba identified as A. astronyxis was isolated from a spinal fluid from a patient with a "non-lethal" meningitis that remitted spontaneo- usly. Free-living amebas are ubiquitous in nature and can easily contaminate any culture (17). However, Acanthamoeba is relatively susceptible to host defense mechanisms and spontaneous remissions may be relatively common. De- tection and study of serum antibodies would permit eval- uation of the significance of such otherwise unsupported cultural results. Free-living amebas, in particular Acanth- amoeba spp., have been isolated from diverse collections of water, like aquariums, swimming pools and lakes, or from the air. Spontaneous infections in animals have been reported. Acanthamoeba were also recovered from the purulent discharge from a human ear. In a series of exten- sive surveys, isolates of free-living amebas were obtained from nasopharyngeal swabs in healthy soldiers but a high- er proportion of positive cultures were observed in sub- jects with nasopharyngeal symptoms (14). Acanthamoe- bic keratitis has been reported with increasing frequency (1). Minor trauma to the coroneal epitheliumappears to be an important factor in the pathogenesis of the ocular infection. The incubation period in Acanthamoeba infection is unknown, but perhaps more than ten days. In CNS infection by Acanthamoeba spp., there is more encephalitis with minimal involvement of the leptomeninges, while in infection due to Naegleria, the leptome- ningitic process is severe and very evident.

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A. J. Martinez, K. Janitsehke: Acanthamoeba, an Opportunistic Microorganism

Cerebral edema and herniations are commonly the leading cause of death. Factors responsible for the spread of the protozoa appear to be the failure of an immune response, the ability of the protozoa to penetrate the natural barriers and the paucity of a glial reaction. The evidence for this appears to be the predominance of nerve cells specialized in sensory and motor function, scanty number of astrocytes and oligodendroglia whose function is plas- ticity and formation of myelin and very few microglia cells or reticular endothelial cells capable of becoming macrophages. It has been demonstrated in experimental animals that the administration of corticosteroids and broad-spectrum antibiotics suppress host defenses and depress host immunity, allowing for the establishment of amebic encephalitis and pneumonitis (15, 16). In addition, acanthamoebic encephalitis was found associated with lupus vul- gads treated with radiation therapy and isoniazide, Hodg- kin's disease treated with immunosuppressive therapy and in chronic alcoholism, malnutrition, cirrhosis of the liver, without immunosuppressive therapy, in apparently "normal" individuals, diabetes mellitus, and dermatolo- gic disorders. Why hyperinfection occurs in certain hosts is not clear. Inadequacy of the immune system appears to be one factor; however, infection can occur without immunosuppression. It will be of interest to evaluate in future patients the

functional and morphological features of the thymus, lymph nodes, and spleen and to rule out an impairment of cell-mediated immune response or other immune defects like IgA deficiency or hypogammaglobulinemia. It is believed that patients with depressed cell-mediated immunity do not produce granulomatous reaction and, therefore, decreased resistance to infections may be ex- pected because of immunologic hyporeactivity. In addition, some of these patients reveal moderate eosinophilia in the CNS. The exact role plasma cells and eosinophils play in the immune response in this infection is unclear. The frequent use of corticosteroids and broad-spectrum antibiotic therapy inhibits or suppresses normal bacterial flora, allowing colonization and overgrowth by fungi and other opportunistic organisms (17). In addition, some damaging effects of the most superficial layer of the mu- cous membranes can be produced by antibiotics, facilitat- ing the penetration of opportunistic pathogens. Opportunistic fungal infections in the ocular adnexa and eyeball can occur by direct external contamination spread from adjacent focus and metastatic from a primary focus elsewhere in the body. Amebic keratitis and endophthal- mitis have been reported (1, 18). The diagnosis of amebic encephalitis is not difficult if the clinical suspicion is high in the proper clinical setting. Of- ten, the disease is diagnosed late in the clinical course or is an unexpected finding at autopsy (19).

Table 3: Comparison of diseases caused by amebas.

Primary disease Diarrhea, abdominal pain, liver Primary amebic meningoencephali- Granulomatous amebic encephali- syndrome abscess, brain abscess tis (PAM) with trophozoites tis (GAE) with trophozoites and

cysts

Epidemiology Oral ingestion of cysts via contami- Recent history of swimming; hot Compromised host. Acquired im- nated food/water summer months munodeficiency syndrome (AIDS)

Portal of entry GI tract Olfactory neuroepithelium Probably lung, skin

Clinical course Variable Acute, fulminant Chronic, slow

Diagnosis Serology, stool examination, tissue CSF examination, tissue exami- Tissue examination: CNS, cornea examination nation skin

CNS involvement Rare (about 200 cases up to De- Rare (one case up to December 31, Very rare (33 cases up to Decembel cember 31, 1984) 1984) 31, 1984)

Ocular involvement None None Possible: Acanthamoeba keratitis (11 cases up to December 31, 1984

Therapy Metronidazole, diiodohydroxyqui- Amphotericin B Miconazole (ketoconazole) noline, tetracycline, emetine, chlo- Possibly sulfadiazine roquine

Differential Ulcerative colitis, cholera, acute CNS: Acute pyogenic (bacterial) CNS: Tuberculous, viral or funga diagnosis appendicitis, diverticulitis leptomeningitis meningitis, brain tumor and ab

scess, ocular: herpes simplex o: fungal keratitis

- CSF High protein, neutrophilic pleocy- Similar to bacterial meningitis but Consistent with viral encephaliti: tosis, no trophozoites sterile, trophozoites active and too- and sterile

tile, cultivation and inoculation in mice

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The search for motile amebas is achieved best by placing one or two drops o f unsta ined CSF, which has been centr ifuged at low speed, on a glass slide, cover slip or a hanging drop and then using an ord inary light microscope with lowered d iaphragm for phase contrast or darkfield. A m e b a s are refractile and they should be different iated f rom lymphocytes , monocytes , po lymorphonuc lea r leukocytes and macrophages . Giemsa or Wright-s ta ined smears o f centr i fuged CSF offer the advantage of showing the characterist ic morpho logy of the ameba which differ- entiates it f rom lymphocytes , erythrocytes and polymor- phonuc lea r leukocytes. Free-living amebas are character ized by a centrally placed, dense nucleolus, su r rounded by a clear nuclear halo and abundant cytoplasm. Usually, Gram-s ta ined smears and bacterial cultures are negative. In tissues, amebas can be easily seen with hematoxyl in and eosin staining. Cysts are be t te r seen with P A S - H . In Table 3 the compar i son and contrast be tween the diseases caused by amebas is de- picted. The t rea tment of acan thamoebic infection with sulfadiazine is p robab ly ineffective, which may reflect the inade-

quacy of the host ' s impaired immune system (15, 20). Ke- toconazole appears to be effective bo th in vitro and in vivo. There is no effective t rea tment available at this t ime for the CNS infection, but ophtha lmic infections like Acan th - amoeba keratitis have been t rea ted with success in some cases with ophthalmic solution o f p ropamidine and dibro- mo propamidine isothionate (13). Awareness o f the opportunist ic nature of A c a n t h a m o e b a infection with its unexpected dissemination and without the presence o f clinical symptoms or epidemiological clues may allow earlier diagnosis o f future cases, advanc- ing the s tudy of the pathogenesis and therapy of this opportunistic infection. In addit ion, because of the world- wide distr ibution of cases of A I D S and its association with multiple opportunis t ic organisms, the possibility of acan thamoebic infection should be considered in the differential diagnosis when t reat ing patients with A I D S .

Acknowledgement

Our sincerest thanks to Miss Karen Perkins for typing and orga- nizing the manuscript.

Literature

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possible pathogenic properties. Zbl. Bakteriol., (Orig. B.) 176 (1982) 160-166.

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13. Shinn, L. E., Hadley, P. B.: Note on the spontaneous contamination of a bacterial culture by an organism resembling Hartmannella castellanii. J. Infect. Dis. 58 (1936) 23-27.

14. Cerva, L., Serbus, C., Skocil, V.: Isolation of limax amoebae from the nasal mucosa of man. Folia Parasitol. (Praha) 20 (1973) 97-103.

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16. Cnlbertson, C. G.: Soil ameba infection. Specific indirect immuno- enzymatic (peroxidase) staining of formalin-fixed paraffin sections. Am. J. Clin. Pathol. 63 (1975) 475-482.

17. Martinez, A. J., Markowitz, S. M., Duma, R. J.: Experimental pneumonitis and encephalitis caused by Acanthamoeba in mice: Pa- thogenesis and ultrastructural features. J. Infect. Dis. 131 (1975) 692-699.

18. Key, S. N., Green, W. R., Willaert, E., Stevens, A.: Keratitis due to Acanthamoeba castellanii. A clinicopathological case report. Arch. Ophthalmol. 98 (1980) 475-479.

19. Willaert, E., Stevens, A. R., Healy, G. R.: Retrospective identification of Acanthamoeba culbertsoni in a case of amebic meningoencephalitis. J. Clin. Pathol. 31 (1978) 717-720.

20. Stevens, A. R., Willaert, E.: Drug sensitivity and resistance of four Acanthamoeba species. Trans. R. Soc. Trop. Med. Hyg. 74 (1980) 806--808.

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1985-Acanthamoeba, an opportunistic microorganism A review

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