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Disease caused by free-living amebae
IIDN1 Volume 8, Number 4, April 1989 Associate Editors
Editor
Paul D. Hoeprich,
Roger G. Finch, FRCP, MRC
MD
Division of Infectious and Immunologic Diseases University of California Davis Medical Center
Ruth M. Lawrence, MD
Path
Department of Microbial Diseases Nottingham City Hospital, U.K.
Veterans Administration Outpatient Clinic Sacramento, California
Larry K. Picketing, MD
Charles W. Stratton, MD
Program in Infectious Diseases and Clinical Microbiology The University of Texas Medical School at Houston
Department of Pathology Vanderbilt University Medical Center Nashville, Tennessee
Disease Caused by Free-Living Amebae
ontent~
Richard J. Duma, MD, PhD Disease Caused by Free-Living Amebae
25
Richard J. Duma, MD, PhD
New Insights into the Diagnosis of AIDS
28
Charles W. Stratton, MD
CASE REPORT
30
Deborah J. Zygmunt, MD COMMENTS ON
CURRENT PUBLICATIONS
31
Department of Medicine, Division of Infectious Diseases Medical College of Virginia Richmond, Virginia
Infections in humans caused by freeliving amebae occur almost exclusively in the central nervous system (CNS) and the eyes. The microrganisms principally responsible belong to the genera Naegleria and Acanthamoeba, of the families Vahlkampfiidae and Acanthamoebidae, respectively. Although the incidence of such infections is low, common° source epidemics, especially with Naegleria spp. frequently occur and result in considerable mortality.
Microbiology
Elsevier 0278-2316/89/$0.00 + 2.20
Free-living amebae are ubiquitous, aerobic protozoa. Both Naegleria spp. and Acanthamoeba spp. are about 10 to 20 ~ in diameter and may take the form of trophozoites or cysts; Naegleria spp. may also transform into flagellates. The trophozoites of Naegleria spp. possess lobose pseudopodia, move in a limax fashion, and are highly motile. In contrast, the trophozoites of Acanthamoeba spp. possess acanthapodia and exhibit sluggish motility.
Characteristically, free-living amebae possess a single nucleus surrounded by a fine nuclear rim and have a large, often centrally placed, karyosome. A single, large contractile vacuole and multiple small food vacuoles may be seen in actively motile and feeding trophozoites. When conditions are difficult for feeding and multiplication, trophozoites may encyst. In addition, trophozoites of Naegleria spp. may transform into highly motile flagellates, a trait useful in their identification. Cysts of Naegleria spp. are round, doublewalled, and possess several pores through which excystation may occur; the cysts of Acanthamoeba spp. are typically stellate, a characteristic that is helpful in detecting them in tissues. The generation time for free-living amebae varies with the incubation temperature and availability of nutrients but is generally around 4 h. Pathogenic Naegleria spp. grow best at 37°C, whereas pathogenic Acanthamoeba spp. grow well at 30°C to 33°C. Growth at 44°C was once
0278-2316 IDINDN 8(4) 25-32, 1989
26
Infectious Diseases Newsletter 8(4) April 1989 thought to be a marker of potentially pathogenic free-living amebae, an observation no longer held valid. Nevertheless, all thermophilic free-living amebae should be considered pathogenic until proved otherwise. In the laboratory, a variety of culture methods and media may be used for primary isolation, eg, amebae may be cultured monaxenically by placing them on a non-nutrient agar seeded with live Escherichia coli or Enterobacter aerogenes. They may also be cultured axenically using artificially enriched media, such as Nelson's for Naegleria spp. and Neff's for Acanthamoeba spp., or they may be cultured in a variety of tissue culture cell lines, particularly those from monkey kidneys. Clinical specimens suspected of containing free-living amebae should be kept or transported at room temperature (23°C) rather than refrigerated~ The addition of saline or amphotericin B to the culture or holding media may be lethal to Naegleria spp. In stained sections of infected tissues or in body liquids, free-living amebae become spherical and may be mistaken for macrophages. On the other hand, living, active macrophages in fresh wet preparations of cerebrospinsal fluid (CSF) may be mistaken for free-living amebae. Such misidentifications are common. However, the presence of cells containing a nucleus with a large karyosome readily distinguishes free-living amebae from mammalian cells. Iron hematoxylin is the best tissue stain for distinguishing the morphologic features of free-living amebae; however, they are readily stained and detected with hematoxylin and eosin (H&E). In tissues infected with Naegleria spp., only trophozoites are found, whereas both cysts and trophozoites may be seen when Acanthamoeba spp. organisms are present.
Diseases
Thus far, only Naegleria spp. are known to produce acute amebic meningitis (AAM), often referred to as primary amebic meningoencephalitis (PAM). Only one species, N. fowleri, has been reported to produce this disease in humans. AAM is an acute, fulminating infection of both the subarachnoid space and the brain. Initially it resembles acute bacterial meningitis in clinical manifestations but very quickly progresses to include aspects of encephalitis and/or a brain abscess. The patients are characteristically healthy young adults or children who give a history of swimming or bathing in warm, fresh water about a week before illness. The disease is usually seasonal, occurring in warm weather when exposure to fresh water is common; AAM may occur in epidemics. Hemorrhagic necrosis of cerebral tissue, particularly of the gray matter, is a hallmark of the disease. The CSF often resembles that seen in acute bacterial meningitis, with a preponderance of neutrophils and an extremely low glucose. However, bacteria are conspicuously absent, and motile trophozoites may be seen. Naegleria fowleri organisms enter the CNS by invading through the olfactory nasal mucosa; they follow the ilia olfactoria into the olfactory bulbs. In the earliest stages of infection, patients may give a history of abnormalities of taste or smell. Although Acanthamoeba spp. may also produce AAM similar to that seen with Naegleria fowleri; they characteristically produce a chronic granulomatous amebic encephalitis (GAE). Several species may be responsible. The clinical picture is generally that of an encephalitis and/ or brain abseess(es)--often located in the midbrain--rather than a meningitis. GAE may develop insidiously
over several weeks and tends to occur in immunodeficient or immunosuppressed patients. Epidemics have not been reported. Acanthameba spp. are believed to enter the CNS via the bloodstream from an extracerebral focus of infection, eg, the skin. Amebae are generally not seen or isolated from the CSF; lymphocytes or mononuclear cells may predominate, and the glucose is often normal or slightly low. The hallmark of the pathology is the presence of a chronic inflammatory response with the formation of granulomas and giant cells. The finding of characteristic stellate cysts (best revealed with Gomori methenamine silver stain) often establishes the diagnosis. Ocular infections with free-living amebae are produced exclusively by Acanthamoeba spp.--most often by
A. polyphaga, A. castellanii, A. rhysodes, and A. culbertsonii. Infections often follow trauma to the cornea, particularly if a foreign body is left in the site of injury. Contact lenses, particularly soft lenses, presumably contaminated with Acanthamoeba spp. prior to insertion, account for most cases. The cornea is prominently involved, and the infection may be suspected when a chronic ulcerating keratitis has not responded to treatment with a variety of antimicrobics and/or hydrocortisone. The infection is most often confused with keratitis caused by herpes simplex viruses; however, with most acanthamoebal infections of the eye, a characteristic radial or "ring" keratoneuritis is seen. Corneal scrapings are often diagnostic, as Acanthamoeba spp. and their cysts can be seen readily in stained preparations and cultured on agar seeded with bacteria. Examination of the ocular lesions with ealcofluor white, a stain for chitin and cellulose (major constituents of cyst walls), is also useful,
NOTE: No respomibitity is assumed by the Publish~ for any injtuy and/or damage to persons ~ ~ as a ~ of ~ ~ , ~ or ~ s e , ~ ~ ~ y use or ~ ~ ~y me~, ~ , inmmetious or ideas contained in the maUninl he~in. No suggested test or Inneednee should be carried out unless, in the reader's judgment, its risk is justified. Becau~ of rapid advances in the medical seienees, we recommend that the independimt vefificainn of diagnoses and di'ug dosages should be made. Discussions, views and rvcommendatinns as to n-~dical procedures, choice of d~gs and dillg timages ate the respomibility of the amhon. Infectious Diseases Newsletter OSSN 0278-2316) is issued monthly in one indexed volume per year by Elsevier Science Publishing Co., inc., 655 Avenue of the Americas, New York, New York 10010. Printed in USA at Hanover, PA 17331. Subscription wice per year: institutions, $138.00; individuals, $80.00. For postage outside the U.S., add $35.00. Second-class postage paid at New York, NY, and at additional mailing offices. Postmaster. Send address changes to Infectious Diseases Newsletter, Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, New York 10010. © 1 9 8 9 E l s e v i e r S c i e n c e P u b l i s h i n g C o . , Inc. 0278-2316/89/$0.00 + 2.20
27 Infectious Diseases Newsletter 8(4) April 1989 but amebae may be difficult to differentiate from yeasts. Serological tests are of no value in diagnosing infections caused by Naegleria spp., because AAM is a rapidly progressive disease with an incubation period that is usually ~<7 days. However, serological studies may be of value in diagnosing subacute or chronic infections caused by Acanthamoeba spp.; acute and convalescent sera must be assayed, because many normal persons carry titers to Acanthamoeba spp.
Treatment To date, only three patients have been documented to have survived AAM caused by N. fowleri, dramatizing the urgency of application of specific therapy. Without treatment, the disease typically procedes to death within 48 h to 72 h after onset of illness. The drug of choice is amphotericin B (AmB); the trophozoites of N. fowleri are exquisitely susceptible to it and are destroyed rapidly, without encystation. One mg/kg body weight should be injected IV within the the first 24 h of disease and repeated daily thereafter. Supplemental administration of AmB by intraventricular injection is hazardous, because the brain is usually markedly swollen and herniation of the brainstem may be precipitated. Other drugs may be given along with AmB, eg, rifampin and/or an imidazole derivative, but their value is questionable. Indeed, the imidazoles may antagonize AmB. The drugs com-
monly used to treat infections caused by Entamoeba histolytica are completely ineffective in both acute amebic encephalitis and granulo-
matous amebic encephalitis; they should not be used. Without treatment, infections of the CNS caused by Acanthamoeba spp. are generally fatal. Abscesses may have to be surgically excised. There are no known anti-acanthamoebal antimicrobics that can penetrate into the CNS in concentrations adequate to destroy amebae. In addition, Acanthamoeba spp. tend to encyst, making total eradication difficult. Sulfadiazine and flucytosine have been used, but their value in the therapy of this disease is very much in doubt. Acanthamoeba spp. are susceptible to polymyxin B and hydroxystilbamidine at concentrations not safely achievable in the CNS. Therefore, the prognosis for curing acanthamoebal disease is extremely poor. Ocular infections caused by Acanthamoeba spp. are best treated both surgically and medically. An occasional patient may be successfully treated with miconazole, ketoconazole, and/or propamidine isethionate. However, a penetrating keratoplasty, or resection of infected tissues with corneal transplantation, is often necessary.
Prevention From studies of mice infected with sublethal inocula of Naegleria spp. or with nonpathogenic strains of Naegleria and from serologic evidence of past infections in humans, a vaccine against naeglerial infections appears possible. However, the application of such a vaccine appears unlikely because of the rarity of AAM. A more practical preventive measure may be the use of nose clips for persons who swim in warm, fresh water.
© 1989 Elsevier Science Publishing Co., Inc. 0278-2316/89/$0.00 + 2.20
Because the epidemiology of Acanthamoeba spp. that infect the CNS is unknown, no preventive measures can be suggested. However, acanthamoebal ocular infections should be preventable by removal of traumatically implanted foreign bodies; moreover, contact lenses should be stored and cleaned only in liquids certified to be free of freeliving amebae.
Bibliography Bush LE, John DT: Intranasal immunization of mice against Naegleriafowleri. J Protozool 35:172-176, 1988.
Duma RJ, Ferrell HW, Nelson EC, et al: Primary amebic meningoencephalitis. New Engl J Med 281:1315-1323, 1969. Duma RJ, Helwig WB, Martinez AJ: Meningocncephalitis and brain abscess due to a free-living amoeba. Ann Int Med 88:468-473, 1978. John DT: Primary amebic meningoencephalitis and the biology of Naegleria fowleri. Ann Rev Microbiol 36:101123, 1982. Martinez, AJ: Acanthamoebiasis and immunosuppression. J Neuropath Exp Neurol 41:548-557, 1982. Moore MB, McCulley JP, Kaufman HE, et al: Radial keratoneuritis as a presenting sign in Acanthamoeba keratitis. Ophthalmology 93:1310-1315, 1986. Parlato C, Davis JC, Cohen E, et al: Acanthamoeba keratitis associated with contact lenses--United States. MMWR 35:405-408, 1986. Seidel JS, Harmatz P, Visvesvara GS, et al: Successful treatment of primary amebic meningoencephalitis. New Engl J Med 306:346-348, 1982. Stevens AR, Shulman ST, Lansen TA, et al: Primary amebic meningoencephalitis: a report of two cases and antibiotic and immunologic studies. J Infect Dis 143:193-199, 1981. Wiley CA, Safrin RE, Davis CE, et al: Acanthamocba meningoencephalitis in a patient with AIDS. J Infect Dis 155:130-133, 1987.
Editor
Paul D. Hoeprich,
Roger G. Finch, FRCP, MRC
MD
Division of Infectious and Immunologic Diseases University of California Davis Medical Center
Ruth M. Lawrence, MD
Path
Department of Microbial Diseases Nottingham City Hospital, U.K.
Veterans Administration Outpatient Clinic Sacramento, California
Larry K. Picketing, MD
Charles W. Stratton, MD
Program in Infectious Diseases and Clinical Microbiology The University of Texas Medical School at Houston
Department of Pathology Vanderbilt University Medical Center Nashville, Tennessee
Disease Caused by Free-Living Amebae
ontent~
Richard J. Duma, MD, PhD Disease Caused by Free-Living Amebae
25
Richard J. Duma, MD, PhD
New Insights into the Diagnosis of AIDS
28
Charles W. Stratton, MD
CASE REPORT
30
Deborah J. Zygmunt, MD COMMENTS ON
CURRENT PUBLICATIONS
31
Department of Medicine, Division of Infectious Diseases Medical College of Virginia Richmond, Virginia
Infections in humans caused by freeliving amebae occur almost exclusively in the central nervous system (CNS) and the eyes. The microrganisms principally responsible belong to the genera Naegleria and Acanthamoeba, of the families Vahlkampfiidae and Acanthamoebidae, respectively. Although the incidence of such infections is low, common° source epidemics, especially with Naegleria spp. frequently occur and result in considerable mortality.
Microbiology
Elsevier 0278-2316/89/$0.00 + 2.20
Free-living amebae are ubiquitous, aerobic protozoa. Both Naegleria spp. and Acanthamoeba spp. are about 10 to 20 ~ in diameter and may take the form of trophozoites or cysts; Naegleria spp. may also transform into flagellates. The trophozoites of Naegleria spp. possess lobose pseudopodia, move in a limax fashion, and are highly motile. In contrast, the trophozoites of Acanthamoeba spp. possess acanthapodia and exhibit sluggish motility.
Characteristically, free-living amebae possess a single nucleus surrounded by a fine nuclear rim and have a large, often centrally placed, karyosome. A single, large contractile vacuole and multiple small food vacuoles may be seen in actively motile and feeding trophozoites. When conditions are difficult for feeding and multiplication, trophozoites may encyst. In addition, trophozoites of Naegleria spp. may transform into highly motile flagellates, a trait useful in their identification. Cysts of Naegleria spp. are round, doublewalled, and possess several pores through which excystation may occur; the cysts of Acanthamoeba spp. are typically stellate, a characteristic that is helpful in detecting them in tissues. The generation time for free-living amebae varies with the incubation temperature and availability of nutrients but is generally around 4 h. Pathogenic Naegleria spp. grow best at 37°C, whereas pathogenic Acanthamoeba spp. grow well at 30°C to 33°C. Growth at 44°C was once
0278-2316 IDINDN 8(4) 25-32, 1989
26
Infectious Diseases Newsletter 8(4) April 1989 thought to be a marker of potentially pathogenic free-living amebae, an observation no longer held valid. Nevertheless, all thermophilic free-living amebae should be considered pathogenic until proved otherwise. In the laboratory, a variety of culture methods and media may be used for primary isolation, eg, amebae may be cultured monaxenically by placing them on a non-nutrient agar seeded with live Escherichia coli or Enterobacter aerogenes. They may also be cultured axenically using artificially enriched media, such as Nelson's for Naegleria spp. and Neff's for Acanthamoeba spp., or they may be cultured in a variety of tissue culture cell lines, particularly those from monkey kidneys. Clinical specimens suspected of containing free-living amebae should be kept or transported at room temperature (23°C) rather than refrigerated~ The addition of saline or amphotericin B to the culture or holding media may be lethal to Naegleria spp. In stained sections of infected tissues or in body liquids, free-living amebae become spherical and may be mistaken for macrophages. On the other hand, living, active macrophages in fresh wet preparations of cerebrospinsal fluid (CSF) may be mistaken for free-living amebae. Such misidentifications are common. However, the presence of cells containing a nucleus with a large karyosome readily distinguishes free-living amebae from mammalian cells. Iron hematoxylin is the best tissue stain for distinguishing the morphologic features of free-living amebae; however, they are readily stained and detected with hematoxylin and eosin (H&E). In tissues infected with Naegleria spp., only trophozoites are found, whereas both cysts and trophozoites may be seen when Acanthamoeba spp. organisms are present.
Diseases
Thus far, only Naegleria spp. are known to produce acute amebic meningitis (AAM), often referred to as primary amebic meningoencephalitis (PAM). Only one species, N. fowleri, has been reported to produce this disease in humans. AAM is an acute, fulminating infection of both the subarachnoid space and the brain. Initially it resembles acute bacterial meningitis in clinical manifestations but very quickly progresses to include aspects of encephalitis and/or a brain abscess. The patients are characteristically healthy young adults or children who give a history of swimming or bathing in warm, fresh water about a week before illness. The disease is usually seasonal, occurring in warm weather when exposure to fresh water is common; AAM may occur in epidemics. Hemorrhagic necrosis of cerebral tissue, particularly of the gray matter, is a hallmark of the disease. The CSF often resembles that seen in acute bacterial meningitis, with a preponderance of neutrophils and an extremely low glucose. However, bacteria are conspicuously absent, and motile trophozoites may be seen. Naegleria fowleri organisms enter the CNS by invading through the olfactory nasal mucosa; they follow the ilia olfactoria into the olfactory bulbs. In the earliest stages of infection, patients may give a history of abnormalities of taste or smell. Although Acanthamoeba spp. may also produce AAM similar to that seen with Naegleria fowleri; they characteristically produce a chronic granulomatous amebic encephalitis (GAE). Several species may be responsible. The clinical picture is generally that of an encephalitis and/ or brain abseess(es)--often located in the midbrain--rather than a meningitis. GAE may develop insidiously
over several weeks and tends to occur in immunodeficient or immunosuppressed patients. Epidemics have not been reported. Acanthameba spp. are believed to enter the CNS via the bloodstream from an extracerebral focus of infection, eg, the skin. Amebae are generally not seen or isolated from the CSF; lymphocytes or mononuclear cells may predominate, and the glucose is often normal or slightly low. The hallmark of the pathology is the presence of a chronic inflammatory response with the formation of granulomas and giant cells. The finding of characteristic stellate cysts (best revealed with Gomori methenamine silver stain) often establishes the diagnosis. Ocular infections with free-living amebae are produced exclusively by Acanthamoeba spp.--most often by
A. polyphaga, A. castellanii, A. rhysodes, and A. culbertsonii. Infections often follow trauma to the cornea, particularly if a foreign body is left in the site of injury. Contact lenses, particularly soft lenses, presumably contaminated with Acanthamoeba spp. prior to insertion, account for most cases. The cornea is prominently involved, and the infection may be suspected when a chronic ulcerating keratitis has not responded to treatment with a variety of antimicrobics and/or hydrocortisone. The infection is most often confused with keratitis caused by herpes simplex viruses; however, with most acanthamoebal infections of the eye, a characteristic radial or "ring" keratoneuritis is seen. Corneal scrapings are often diagnostic, as Acanthamoeba spp. and their cysts can be seen readily in stained preparations and cultured on agar seeded with bacteria. Examination of the ocular lesions with ealcofluor white, a stain for chitin and cellulose (major constituents of cyst walls), is also useful,
NOTE: No respomibitity is assumed by the Publish~ for any injtuy and/or damage to persons ~ ~ as a ~ of ~ ~ , ~ or ~ s e , ~ ~ ~ y use or ~ ~ ~y me~, ~ , inmmetious or ideas contained in the maUninl he~in. No suggested test or Inneednee should be carried out unless, in the reader's judgment, its risk is justified. Becau~ of rapid advances in the medical seienees, we recommend that the independimt vefificainn of diagnoses and di'ug dosages should be made. Discussions, views and rvcommendatinns as to n-~dical procedures, choice of d~gs and dillg timages ate the respomibility of the amhon. Infectious Diseases Newsletter OSSN 0278-2316) is issued monthly in one indexed volume per year by Elsevier Science Publishing Co., inc., 655 Avenue of the Americas, New York, New York 10010. Printed in USA at Hanover, PA 17331. Subscription wice per year: institutions, $138.00; individuals, $80.00. For postage outside the U.S., add $35.00. Second-class postage paid at New York, NY, and at additional mailing offices. Postmaster. Send address changes to Infectious Diseases Newsletter, Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, New York 10010. © 1 9 8 9 E l s e v i e r S c i e n c e P u b l i s h i n g C o . , Inc. 0278-2316/89/$0.00 + 2.20
27 Infectious Diseases Newsletter 8(4) April 1989 but amebae may be difficult to differentiate from yeasts. Serological tests are of no value in diagnosing infections caused by Naegleria spp., because AAM is a rapidly progressive disease with an incubation period that is usually ~<7 days. However, serological studies may be of value in diagnosing subacute or chronic infections caused by Acanthamoeba spp.; acute and convalescent sera must be assayed, because many normal persons carry titers to Acanthamoeba spp.
Treatment To date, only three patients have been documented to have survived AAM caused by N. fowleri, dramatizing the urgency of application of specific therapy. Without treatment, the disease typically procedes to death within 48 h to 72 h after onset of illness. The drug of choice is amphotericin B (AmB); the trophozoites of N. fowleri are exquisitely susceptible to it and are destroyed rapidly, without encystation. One mg/kg body weight should be injected IV within the the first 24 h of disease and repeated daily thereafter. Supplemental administration of AmB by intraventricular injection is hazardous, because the brain is usually markedly swollen and herniation of the brainstem may be precipitated. Other drugs may be given along with AmB, eg, rifampin and/or an imidazole derivative, but their value is questionable. Indeed, the imidazoles may antagonize AmB. The drugs com-
monly used to treat infections caused by Entamoeba histolytica are completely ineffective in both acute amebic encephalitis and granulo-
matous amebic encephalitis; they should not be used. Without treatment, infections of the CNS caused by Acanthamoeba spp. are generally fatal. Abscesses may have to be surgically excised. There are no known anti-acanthamoebal antimicrobics that can penetrate into the CNS in concentrations adequate to destroy amebae. In addition, Acanthamoeba spp. tend to encyst, making total eradication difficult. Sulfadiazine and flucytosine have been used, but their value in the therapy of this disease is very much in doubt. Acanthamoeba spp. are susceptible to polymyxin B and hydroxystilbamidine at concentrations not safely achievable in the CNS. Therefore, the prognosis for curing acanthamoebal disease is extremely poor. Ocular infections caused by Acanthamoeba spp. are best treated both surgically and medically. An occasional patient may be successfully treated with miconazole, ketoconazole, and/or propamidine isethionate. However, a penetrating keratoplasty, or resection of infected tissues with corneal transplantation, is often necessary.
Prevention From studies of mice infected with sublethal inocula of Naegleria spp. or with nonpathogenic strains of Naegleria and from serologic evidence of past infections in humans, a vaccine against naeglerial infections appears possible. However, the application of such a vaccine appears unlikely because of the rarity of AAM. A more practical preventive measure may be the use of nose clips for persons who swim in warm, fresh water.
© 1989 Elsevier Science Publishing Co., Inc. 0278-2316/89/$0.00 + 2.20
Because the epidemiology of Acanthamoeba spp. that infect the CNS is unknown, no preventive measures can be suggested. However, acanthamoebal ocular infections should be preventable by removal of traumatically implanted foreign bodies; moreover, contact lenses should be stored and cleaned only in liquids certified to be free of freeliving amebae.
Bibliography Bush LE, John DT: Intranasal immunization of mice against Naegleriafowleri. J Protozool 35:172-176, 1988.
Duma RJ, Ferrell HW, Nelson EC, et al: Primary amebic meningoencephalitis. New Engl J Med 281:1315-1323, 1969. Duma RJ, Helwig WB, Martinez AJ: Meningocncephalitis and brain abscess due to a free-living amoeba. Ann Int Med 88:468-473, 1978. John DT: Primary amebic meningoencephalitis and the biology of Naegleria fowleri. Ann Rev Microbiol 36:101123, 1982. Martinez, AJ: Acanthamoebiasis and immunosuppression. J Neuropath Exp Neurol 41:548-557, 1982. Moore MB, McCulley JP, Kaufman HE, et al: Radial keratoneuritis as a presenting sign in Acanthamoeba keratitis. Ophthalmology 93:1310-1315, 1986. Parlato C, Davis JC, Cohen E, et al: Acanthamoeba keratitis associated with contact lenses--United States. MMWR 35:405-408, 1986. Seidel JS, Harmatz P, Visvesvara GS, et al: Successful treatment of primary amebic meningoencephalitis. New Engl J Med 306:346-348, 1982. Stevens AR, Shulman ST, Lansen TA, et al: Primary amebic meningoencephalitis: a report of two cases and antibiotic and immunologic studies. J Infect Dis 143:193-199, 1981. Wiley CA, Safrin RE, Davis CE, et al: Acanthamocba meningoencephalitis in a patient with AIDS. J Infect Dis 155:130-133, 1987.