NON-NAEGLERIA AMEBIC MENINGOENCEPHALITIS RICHARD J. DUMA, M.D., PH.D. RICHMOND, VIRGINIA
In 1958, the potential for free-living amebas to produce disease in Man was fiLrst reported in the journal Science by Culbertson, Smith, and Minner (1), after a species of Acanthamoeba, thought initially to be a vaccine strain of poliomyelitis virus, was found "contaminating" a batch of polio vaccine. This ameba produced a cytopathic effect within 48 hours when inoculated onto primary monkey kidney cells and proved to be rapidly fatal when inoculated into mice or monkeys, producing a paralytic disease characterized by hemorrhagic necrosis of the cerebral cortex. In 1965, the discovery of natural infections in humans due to free-living amebas occurred when Fowler and Carter (2) in South Australia reported several autopsied cases of remarkably similar but previously undescribed meningoencephalitis. Noted within cerebral tissues were large numbers of peculiar, magenta-colored cells, each containing a single large karyosome and what appeared to be a contractile vacuole. Although these cells had been called "gitter cells" or macrophages by others, they were instead amebas. Because of Culbertson's work, Fowler and Carter believed the organism responsible for the disease they described was an Acanthamoeba; however, subsequently it was shown that the organism was instead Naegleria, a related, free-living amebo-flagellate, but distinctly different from Acanthamoeba. Soon afterwards, the term Primary Amebic Meningoencephalitis or PAM was coined to distinguish a disease process characterized by direct invasion of the central nervous system (CNS) by aerobic, free-living amebas from that produced by Entamoeba histolytica, an anaerobic, coprozoic parasite, which secondarily infects the CNS from a distant site, usually located in the lung and/or liver. PAM produced by Naegleria is a relatively well known entity, both to physicians and public health officials, because the source is fresh water, and infection is usually acquired from swimming in public lakes or pools; or because the organism is known to cause epidemics; or because the infections due to it have been so dramatically devastating and rapidly fatal; or most likely because of all of the aforementioned. Be that as it may, the object of this presentation is to discuss and compare the clinical, pathological and epidemiological features of a less-well understood entity, Division of Infectious Diseases, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298. 65
66
RICHARD J. DUMA
one that I believe is frequently missed by clinicians and neuropathologists alike, and which I believe is becoming of increasing importance, namely, PAM due to non-Naegleria free-living amebas (as I choose to call them), most of which are probably Acanthamoeba. To date, about 80 cases of PAM due to Naegleria have been recognized (about half in the USA); while only about 17 to 22 cases, depending on one's criteria, of nonNaegleria PAM have been reported (Table 1). It should be emphasized at the outset that each case of PAM putatively due to Acanthamoeba, or alluded to as Acanthamoeba sp., have been diagnosed either by immunological methods (usually indirect immunofluorescence of fixed tissue sections) or by the presence of what are believed to be the presence of "characteristic" trophozoites and cyst forms. The former criterion, immunofluorescence, appears to be reliable and highly specific; however the latter, the presence of characteristic trophozoite or cyst forms, is tenuous. To date, in no instance of human PAM have Acanthamoeba or non-Naegleria, free-living amebas been isolated and cultured from the CNS (most cases having been diagnosed postmortem after tissue fixation when amebas are no longer viable and cultureable); whereas, Naegleria, which has been associated with a well described, characteristic infectious syndrome, has been cultured from cerebrospinal fluid or brain tissue on many occasionts both pre- and postmortem (before tissue fixation). A word or two should be offered concerning the classification and nomenclature of free-living amebas, even though these subjects are still TABLE I Primary Meningoencephalitis due to Free-Living Amebas Other than Naegleria Age & Sex
Year
22 M 6F 5M 16 M 59 M 23 M 57 M 58 M 20 mo. M 7M 47 F 26 M Adult M 30 M 56 M 20 M 11 F
1943 N. Guinea 1955 Arizona 1958 Korea 1959 New York 1964 Texas 1969 Uganda 1970? New York 1971 Pennsylvania 1972 New York 1973 Texas 1974 Virginia 1975? Venezuela 1975 Zambia 1975 New York 1975 Louisiana 1977 Peru 1978 New York
Location
Organism
Reference
Iodamoeba butschlii L butschlii Acanthamoeba Acanthamoeba Acanthamoeba Unknown Acanthamoeba Acanthamoeba Unknown Acanthamoeba Unknown A. culbertsoni Acanthamoeba Acanthamoeba? Acanthamoeba A. castellanii Acanthamoeba
Derrick (4) Kernohan et al (5) Ringsted et al (6) Jager & Stamm (7) Patras & Andujar (8) Grundy & Blowers (9) Kenny (10) Robert & Rorke (11) Napolitano et al (12) Sotelo-Avila (13) Duma (14) Martinez et al (15) Bhagwandeen et al (16) Napolitano et al (12) Hoffman et al (13) Martinez et al (15) Powers (17)
NON-NAEGLERIA AMEBIC MENINGOENCEPHALITIS
67
under some debate. N. fowleri (the only identified species pathogenic for Man) is variously referred to in the literature as N. aerobia and N. invades; and some investigators, e.g., Singh, do not accept the genus Acanthamoeba at all, preferring to classify these organisms under the genus Hartmannella. Thus, in the literature Acanthamoeba and Hartmannella are often used to describe the same organism. To date the best and most accepted classification of free-living amebas is that of Page (3), who has incorporated features of nuclear structures; mitosis and division patterns; cytological features including motility; biochemical; and immunological characteristics. The fact that this classification does not incorporate pathogenicity as a distinguishing feature is fortunate and worthwhile, as all free-living amebas should be considered potentially pathogenic until proven otherwise. In Page's classification, three families in the order Amoebida are recognized as containing amebas pathogenic for man: Entamoebidae, Acanthamoebidae, and Vahlkampfiidae. Only the latter two families contain free-living amebas. Human infections due to amebas classified by Page as Hartmannella have not been recognized thus far. It is important to appreciate that the free-living amebas considered pathogenic for Man are highly aerobic; may be found worldwide in soil but more importantly in water; have no known animal reservoir; multiply rapidly; are hardy and versatile in their forms; are small (generally about 12 to 40 microns in diameter); and that potentially one ameba may infect a person if circumstances permit. Figure 1 illustrates graphically some of the salient morphologic features of several of the known pathogenic species of free-living amebas and compares them with E. histolytica. Note the single large karyosome of Naegleria and Acanthamoeba, as distinguished from the tiny centrally located karyosome of the parasite E. histolytica. Also, note the flagellate form of Naegleria. Naegleria can transform readily into a flagellate when environmental conditions dictate and then back again into a trophozoite form for feeding and multiplying. Note the cyst forms, which for Naegleria are characteristically round, double-walled and may contain pores; but which for Acanthamoeba are stellate, wrinkled and without pores. The cysts of both the free-living amebas are uninucleate as compared to the multinucleated ones of E. histolytica. Trophozoites in the living condition are distinguished by their motility and the morphology of their pseudopods; Naegleria is highly motile while Acanthamoeba usually is sluggish. In fixed tissues after staining, it should be appreciated that all three of the amebas illustrated round up and are indistinguishable in gross appearance and shape. Immunologically, the genera Acanthamoeba and Naegleria can be easily distinguished, both in terms of surface and solubilized antigens. However,
68
RICHARD J. DUMA
ENTAMOEBA HISTOLYTICA / s
I
MULTINUCLEATED CYST
TROPHOZOITE
NAEGLERIA FOWLERI
FIG. 1. Note tiny centrally located karyosome and collection of chromatin about the nuclear rim for E. histolytica as compared to the large karyosomes and fine nuclear rims for A. castellanii and N. fowleri. Also note differences between the cyst forms and the flagellate stage seen with Naegleria. Morphologic differences between the trophozoite forms of Acanthamoeba and Naegleria are not always easily defined, and after fixation the two trophozoites are virtually indistinguishable. (Cysts are approximately 16 micra in diameter. Sizes of trophozoites and the flaggelate, relative to cyst sizes, are appropriately drawn.)
species differentiation within a genus is more difficult immunologically because of antigenic similarities. To date, about 17 cases of well-documented, non-Naegleria infections have been published (4-17) (Table 1). The number has been increasing steadily, year by year, since the first reported cases. Although this increase may be more apparent than real and may be related to increasing interest in the subject, it is nevertheless impressive. Over half of the cases have been from the United States, although a wide variety of countries have been represented. In 12 of 17 cases, Acanthamoeba species were
NON-NAEGLERIA AMEBIC MENINGOENCEPHALITIS
69
TABLE 2
Comparison of Naegleria (N) and Non-Naegleria (N-N) Primary Amebic Meningoencephalitis Factors
N-N
N
Age Sex Prior Health
Child or young adult Males 3:2 Excellent
Epidemiology
History of recent intimate contact
Any age Males 5:1 Often underlying diseases present (e.g., cirrhosis, Hodgkin's, etc.) Unknown
with fresh water (e.g.. swimming) 5-7 davs
Unikiowni
Incubation period P'ortal of entry
Invasion route to CNS Clinical picture Clinical course l'reatment
Pathology
l'rotozoology
Olfactory mucosa
Olfactorv nerves Resembles "acuite pyogenic meningitis" Rapidly fatal (2-3 days) Amphotericin B Hemorrhagic necrosis (acute neutrophilic) response with involvement of frontal, tenmporal, &/or cerebellar and inivariably olfactory lobes
Onlv trophs pr.-;Pent Amebas seen li! inad isolated fro.,. CSF
Skin, prostate, uterus, eve, lung, ear, olfactory mucosa, and perhaps others Usually hematogenous, but also on occasion via olfactory nerve Resembles brain abscess(es) Fatal but usuallv chronic with occasional course acute None Hemorrhagic necrosis (chronic inflammatory cells, reparative gliosis and granulomas) Mid-line structures, but olfactory usually spared Both trophs and cvsts present No amebas seen in or isolated from CSF
believed to be the responsil)le pathogen, and indeed most likely were. For two of the earlier cases Iodutmoeb'i bultsch lii was believed responsible, but this could well be incorrect. Ages of patients have ranged from 20 months to 59 years. Males hlave predominated strikingly (Table 2). In comparing Naegleria to non-Naegleria PAM (Table 2), one finds not only a general difference in age an(d sex of the patient, but also differences in a variety of other factors. Patients with non-Naegleria infections often have seriou-s predisposing. underlying diseases, rendering them immunocompromise(1; and the source of their pathogen is obscure, except that occasionally such amebas can be isolated from the throats and noses of "normal" people (18. 19) an(d perhaps even are "carried" there. Thus, endogenously acquire(l infe(tions are certainly possible. The incubation period for- non-Naegleria infection is unclear, and the l)ortal of enitrv may vary, v,"ahereas 1)oth oi these aspects are well under-
70
RICHARD J. DUMA
stood for Naegleria PAM. Concomitant infections other than in the CNS have been reported in the Acanthamoeba PAM, e.g., in the prostate (15), uterus (15), skin (9), eye (15); and intestine. Often these have occurred before appearances of CNS infections, suggesting secondary hematogenous spread to the CNS. Although little clinical evidence exists to support invasion along the olfactory paths, experimental infections would suggest that occasionally this route also may be followed (20). The clinical picture of non-Naegleria infections is generally subacute or chronic, and most resemble that seen with a brain abscess or abscesses, whereas it is acute and fulminant for PAM due to Naegleria. There is no effective treatment known for Acanthamoeba infections, while amphotericin B, the antifungal agent, is highly effective against Naegleria. For non-Naegleria infections, involvement of the CNS is often diffuse and includes many midline structures, e.g., pons, medulla, caudate nucleus, and thalamus. On the other hand Naegleria invariably involves the olfactory lobe, and often confines its spread locally to temporal, frontal, and occasionally cerebellar lobes. The CSF in non-Naegleria infections usually contains a preponderance of round cells, normal or slightly low sugar, modest elevation of proteins, and amebas have not been seen or cultured from it. On the other hand, in Naegleria infections, amebas have often been seen and cultured from the CSF, the cellular reaction is predominantly neutrophilic, and the sugar is often markedly low. In conclusion: 1. PAM due to non-Naegleria organisms probably is most often due to Acanthamoeba sps.; however, a variety of free-living amebas may be involved. 2. The clinical picture of such infections is generally chronic (but occasionally may be acute), resembles that of a brain abscess(es), and more commonly occurrs in the compromised host. 3. Organs other than the brain may be involved, as infection of the CNS appears to result from hematogenous spread from a distant primary site. 4. The disease to our knowledge is invariably fatal, and medical treatment is not yet available. 5. The epidemiology is unclear, although such amebas may be found worldwide, are highly ubiquitous in Nature, are commonly found in soil and water, and even may be found in the normal upper respiratory passages of man. 6. Prevention is not employed because we don't know what to do, but natural immunity probably exists. Public health measures to control or prevent such infections may be impossible or impractical, because of the diffuseness of the problem and the variety, large numbers, and widespread natural distribution of the organisms responsible.
NON-NAEGLERIA AMEBIC MENINGOENCEPHALITIS
71
REFERENCES 1. CULBERTSON, C. G., SMITH, J. AND MINNER, J.: Acanthamoeba: observations on animal pathogenicity. Science 127: 1506, 1958. 2. FOWLER, M. AND CARTER, R. F.: Acute pyogenic meningitis probably due to Acantha-
moeba sp. Brit. Med. J. 2: 740, 1965. 3. PAGE, F. C.: A revised classification of the Gymnamoebia (Protozoa Sarcodina). Zool. J. Linnean Soc. 58: 61, 1976. 4. DERRICK, E. H.: A fatal case of generalized amoebiasis due to a protozoan closely resembling, if not identical with, Iodamoeba butschlii. Trans. Roy. Soc. Trop. Med. Hyg. 42: 191, 1948. 5. KERNOHAN, J. W., MAGETH, T. B. AND SCHLOSS, G. T.: Granuloma of brain probably due to Endolimax williamsi (Iodamoeba butschlii). Arch. Path. 70: 576, 1960. 6. RINGSTED, J., JAGER, B. V., SUK, D., AND VISVESVARA, G. S.: Probable Acanthamoeba Meningoencephalitis in a Korean Child. Amer. J. Clin. Path. 66: 723, 1976. 7. JAGER, B. V. AND STAMM, W. P.: Brain abscesses caused by free-living amoeba probably of the genus Hartmannella in a patient with Hodgkin's disease. Lancet 2: 1343, 1972. 8. PATRAS, D. AND ANDUJAR, J. J.: Meningo-encephalitis due to Hartmannella (Acanthamoeba). Amer. J. Clin. Path. 46: 226, 1966. 9. GRUNDY, R. AND BLOWERS, R.: A case of primary amoebic meningoencephalitis treated with chloroquine. E. Afr. Med. J. 47: 153, 1970. 10. KENNY, M.: The micro-Kolmer complement fixation test in routine screening for soil ameba infection. Hlth. Lab. Sci. 8: 5, 1971. 11. ROBERT, V. B. AND RORKE, L. B.: Primary amebic encephalitis, probably from Acanthamoeba. Ann. Intern. Med. 79: 174, 1973. 12. NAPOLITANO, J. J., GRILLO, R. S., AND GAMBLE, H. R.: Primary Amebic Meningoencephalitis. New York State J. Med. 78: 1883, 1978. 13. HOFFMAN, E. O., GARCIA, C., LUNSETH, J., McGARRY, P., AND COOVER, J.: A case of primary amebic meningoencephalitis. Light and electron microscopy, and immunohistologic studies. Amer. J. Trop. Med. Hyg. 27: 29, 1978. 14. DUMA, R. J., HELWIG, W. B., AND MARTINEZ, A. J.: Meningoencephalitis and brain abscess due to a free-living amoeba. Ann. Intern. Med. 88: 468, 1978. 15. MARTINEZ, A. J., SOTELO-AVILA, C., GARCIA-TAMAYO, J., MORON, J. T., WILLAERT, E., AND STAMM, W. P.: Meningoencephalitis due to acanthamoeba sp. Pathogenesis and clinico-pathological study. Acta Neuropath. 37: 183, 1977. 16. BHAGWANDEEN, S. B., CARTER, R. F., NAIK, K. G., AND LEvIrn, D.: A case of hartmannellid amebic meningoencephalitis in Zambia. Amer. J. Clin. Path. 63: 483, 1975. 17. POWERS, P.: Primary amebic meningoencephalitis-New York. Morbidity and Mortality Weekly Report. 27: 343, 1978. 18. WANG, S. S. AND FELDMAN, H. A.: Isolation of Hartmannella species from human throats. New Eng. J. Med. 277: 1174, 1967. 19. CHANG, S. L., HEALY, G. R., MCCABE, L., SHUMAKER, J. B., AND SCHLUTZ, M. G.: A strain of pathogenic Naegleria isolated from a human nasal swab. HIth Lab. Sci. 12: 1, 1975. 20. MARTINEZ, A. J., MARKOWITZ, S. M., AND DUMA, R. J.: Experimental pneumonitis and encephalitis caused by Acanthamoeba in mice: pathogenesis and ultrastructural features. J. Infec. Dis. 131: 692, 1975.
DISCUSSION DR. HOOK (Charlottesville): Thank you for a most interesting paper. Do these amoebas
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RICHARD J. DUMA
produce infection in animals and it so, do vou find impressive tropism for the central nervous system? DR. DUMA (Richmond): Yes, they do. 'l ropism for the CNS certainly exists for Naegleria, as naeglerial infections seem to be linited to the CNS. One can produce infections with N. fowleri in a wide variety of inoculationi routes. With Acanthamoebas, CNS tropism appears to be less certain, although it appears that every species of Acanthamoeba (and there are many) carries the potential to produce infection. Some common animals infected by freeliving amebas are the marine animals; indeed, sizeable epidemics have occurred in mollusks, oysters, and crabs. Also, natural infections in several mammals have been obser',ved incidentally, although no systematic search for such infections has been made. DR. WITHAM (Augusta): Is it known what the incidence of infections from ponds might be, say fresh water ponds in Virginia, from both the Naegleria and non-Naegleria amoebae'? Also, does chlorine kill them in your swimming pool? DR. DUMA: No cases of PAM has-e been acquired from swimming pools in this countrY (USA), especially not chlorinated pools. In many of the European countries where heate(d pools exist, a number of cases and even tpidemics have occurred; for example, in Belgiulml and in Czechoslovakia. In Virginia where recentlv we have completed a 3 vear study of the ecologv of several lakes surrounding the city of Richmond (and I know Dr. Hook won't like to hear this, since he lives nearby) pathogenic Naegleria have been isolated on numerous occasions from one of the lakes. This lake is interesting because it serves as a coolant for a nearby electrical power plant; and a
In 1958, the potential for free-living amebas to produce disease in Man was fiLrst reported in the journal Science by Culbertson, Smith, and Minner (1), after a species of Acanthamoeba, thought initially to be a vaccine strain of poliomyelitis virus, was found "contaminating" a batch of polio vaccine. This ameba produced a cytopathic effect within 48 hours when inoculated onto primary monkey kidney cells and proved to be rapidly fatal when inoculated into mice or monkeys, producing a paralytic disease characterized by hemorrhagic necrosis of the cerebral cortex. In 1965, the discovery of natural infections in humans due to free-living amebas occurred when Fowler and Carter (2) in South Australia reported several autopsied cases of remarkably similar but previously undescribed meningoencephalitis. Noted within cerebral tissues were large numbers of peculiar, magenta-colored cells, each containing a single large karyosome and what appeared to be a contractile vacuole. Although these cells had been called "gitter cells" or macrophages by others, they were instead amebas. Because of Culbertson's work, Fowler and Carter believed the organism responsible for the disease they described was an Acanthamoeba; however, subsequently it was shown that the organism was instead Naegleria, a related, free-living amebo-flagellate, but distinctly different from Acanthamoeba. Soon afterwards, the term Primary Amebic Meningoencephalitis or PAM was coined to distinguish a disease process characterized by direct invasion of the central nervous system (CNS) by aerobic, free-living amebas from that produced by Entamoeba histolytica, an anaerobic, coprozoic parasite, which secondarily infects the CNS from a distant site, usually located in the lung and/or liver. PAM produced by Naegleria is a relatively well known entity, both to physicians and public health officials, because the source is fresh water, and infection is usually acquired from swimming in public lakes or pools; or because the organism is known to cause epidemics; or because the infections due to it have been so dramatically devastating and rapidly fatal; or most likely because of all of the aforementioned. Be that as it may, the object of this presentation is to discuss and compare the clinical, pathological and epidemiological features of a less-well understood entity, Division of Infectious Diseases, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298. 65
66
RICHARD J. DUMA
one that I believe is frequently missed by clinicians and neuropathologists alike, and which I believe is becoming of increasing importance, namely, PAM due to non-Naegleria free-living amebas (as I choose to call them), most of which are probably Acanthamoeba. To date, about 80 cases of PAM due to Naegleria have been recognized (about half in the USA); while only about 17 to 22 cases, depending on one's criteria, of nonNaegleria PAM have been reported (Table 1). It should be emphasized at the outset that each case of PAM putatively due to Acanthamoeba, or alluded to as Acanthamoeba sp., have been diagnosed either by immunological methods (usually indirect immunofluorescence of fixed tissue sections) or by the presence of what are believed to be the presence of "characteristic" trophozoites and cyst forms. The former criterion, immunofluorescence, appears to be reliable and highly specific; however the latter, the presence of characteristic trophozoite or cyst forms, is tenuous. To date, in no instance of human PAM have Acanthamoeba or non-Naegleria, free-living amebas been isolated and cultured from the CNS (most cases having been diagnosed postmortem after tissue fixation when amebas are no longer viable and cultureable); whereas, Naegleria, which has been associated with a well described, characteristic infectious syndrome, has been cultured from cerebrospinal fluid or brain tissue on many occasionts both pre- and postmortem (before tissue fixation). A word or two should be offered concerning the classification and nomenclature of free-living amebas, even though these subjects are still TABLE I Primary Meningoencephalitis due to Free-Living Amebas Other than Naegleria Age & Sex
Year
22 M 6F 5M 16 M 59 M 23 M 57 M 58 M 20 mo. M 7M 47 F 26 M Adult M 30 M 56 M 20 M 11 F
1943 N. Guinea 1955 Arizona 1958 Korea 1959 New York 1964 Texas 1969 Uganda 1970? New York 1971 Pennsylvania 1972 New York 1973 Texas 1974 Virginia 1975? Venezuela 1975 Zambia 1975 New York 1975 Louisiana 1977 Peru 1978 New York
Location
Organism
Reference
Iodamoeba butschlii L butschlii Acanthamoeba Acanthamoeba Acanthamoeba Unknown Acanthamoeba Acanthamoeba Unknown Acanthamoeba Unknown A. culbertsoni Acanthamoeba Acanthamoeba? Acanthamoeba A. castellanii Acanthamoeba
Derrick (4) Kernohan et al (5) Ringsted et al (6) Jager & Stamm (7) Patras & Andujar (8) Grundy & Blowers (9) Kenny (10) Robert & Rorke (11) Napolitano et al (12) Sotelo-Avila (13) Duma (14) Martinez et al (15) Bhagwandeen et al (16) Napolitano et al (12) Hoffman et al (13) Martinez et al (15) Powers (17)
NON-NAEGLERIA AMEBIC MENINGOENCEPHALITIS
67
under some debate. N. fowleri (the only identified species pathogenic for Man) is variously referred to in the literature as N. aerobia and N. invades; and some investigators, e.g., Singh, do not accept the genus Acanthamoeba at all, preferring to classify these organisms under the genus Hartmannella. Thus, in the literature Acanthamoeba and Hartmannella are often used to describe the same organism. To date the best and most accepted classification of free-living amebas is that of Page (3), who has incorporated features of nuclear structures; mitosis and division patterns; cytological features including motility; biochemical; and immunological characteristics. The fact that this classification does not incorporate pathogenicity as a distinguishing feature is fortunate and worthwhile, as all free-living amebas should be considered potentially pathogenic until proven otherwise. In Page's classification, three families in the order Amoebida are recognized as containing amebas pathogenic for man: Entamoebidae, Acanthamoebidae, and Vahlkampfiidae. Only the latter two families contain free-living amebas. Human infections due to amebas classified by Page as Hartmannella have not been recognized thus far. It is important to appreciate that the free-living amebas considered pathogenic for Man are highly aerobic; may be found worldwide in soil but more importantly in water; have no known animal reservoir; multiply rapidly; are hardy and versatile in their forms; are small (generally about 12 to 40 microns in diameter); and that potentially one ameba may infect a person if circumstances permit. Figure 1 illustrates graphically some of the salient morphologic features of several of the known pathogenic species of free-living amebas and compares them with E. histolytica. Note the single large karyosome of Naegleria and Acanthamoeba, as distinguished from the tiny centrally located karyosome of the parasite E. histolytica. Also, note the flagellate form of Naegleria. Naegleria can transform readily into a flagellate when environmental conditions dictate and then back again into a trophozoite form for feeding and multiplying. Note the cyst forms, which for Naegleria are characteristically round, double-walled and may contain pores; but which for Acanthamoeba are stellate, wrinkled and without pores. The cysts of both the free-living amebas are uninucleate as compared to the multinucleated ones of E. histolytica. Trophozoites in the living condition are distinguished by their motility and the morphology of their pseudopods; Naegleria is highly motile while Acanthamoeba usually is sluggish. In fixed tissues after staining, it should be appreciated that all three of the amebas illustrated round up and are indistinguishable in gross appearance and shape. Immunologically, the genera Acanthamoeba and Naegleria can be easily distinguished, both in terms of surface and solubilized antigens. However,
68
RICHARD J. DUMA
ENTAMOEBA HISTOLYTICA / s
I
MULTINUCLEATED CYST
TROPHOZOITE
NAEGLERIA FOWLERI
FIG. 1. Note tiny centrally located karyosome and collection of chromatin about the nuclear rim for E. histolytica as compared to the large karyosomes and fine nuclear rims for A. castellanii and N. fowleri. Also note differences between the cyst forms and the flagellate stage seen with Naegleria. Morphologic differences between the trophozoite forms of Acanthamoeba and Naegleria are not always easily defined, and after fixation the two trophozoites are virtually indistinguishable. (Cysts are approximately 16 micra in diameter. Sizes of trophozoites and the flaggelate, relative to cyst sizes, are appropriately drawn.)
species differentiation within a genus is more difficult immunologically because of antigenic similarities. To date, about 17 cases of well-documented, non-Naegleria infections have been published (4-17) (Table 1). The number has been increasing steadily, year by year, since the first reported cases. Although this increase may be more apparent than real and may be related to increasing interest in the subject, it is nevertheless impressive. Over half of the cases have been from the United States, although a wide variety of countries have been represented. In 12 of 17 cases, Acanthamoeba species were
NON-NAEGLERIA AMEBIC MENINGOENCEPHALITIS
69
TABLE 2
Comparison of Naegleria (N) and Non-Naegleria (N-N) Primary Amebic Meningoencephalitis Factors
N-N
N
Age Sex Prior Health
Child or young adult Males 3:2 Excellent
Epidemiology
History of recent intimate contact
Any age Males 5:1 Often underlying diseases present (e.g., cirrhosis, Hodgkin's, etc.) Unknown
with fresh water (e.g.. swimming) 5-7 davs
Unikiowni
Incubation period P'ortal of entry
Invasion route to CNS Clinical picture Clinical course l'reatment
Pathology
l'rotozoology
Olfactory mucosa
Olfactorv nerves Resembles "acuite pyogenic meningitis" Rapidly fatal (2-3 days) Amphotericin B Hemorrhagic necrosis (acute neutrophilic) response with involvement of frontal, tenmporal, &/or cerebellar and inivariably olfactory lobes
Onlv trophs pr.-;Pent Amebas seen li! inad isolated fro.,. CSF
Skin, prostate, uterus, eve, lung, ear, olfactory mucosa, and perhaps others Usually hematogenous, but also on occasion via olfactory nerve Resembles brain abscess(es) Fatal but usuallv chronic with occasional course acute None Hemorrhagic necrosis (chronic inflammatory cells, reparative gliosis and granulomas) Mid-line structures, but olfactory usually spared Both trophs and cvsts present No amebas seen in or isolated from CSF
believed to be the responsil)le pathogen, and indeed most likely were. For two of the earlier cases Iodutmoeb'i bultsch lii was believed responsible, but this could well be incorrect. Ages of patients have ranged from 20 months to 59 years. Males hlave predominated strikingly (Table 2). In comparing Naegleria to non-Naegleria PAM (Table 2), one finds not only a general difference in age an(d sex of the patient, but also differences in a variety of other factors. Patients with non-Naegleria infections often have seriou-s predisposing. underlying diseases, rendering them immunocompromise(1; and the source of their pathogen is obscure, except that occasionally such amebas can be isolated from the throats and noses of "normal" people (18. 19) an(d perhaps even are "carried" there. Thus, endogenously acquire(l infe(tions are certainly possible. The incubation period for- non-Naegleria infection is unclear, and the l)ortal of enitrv may vary, v,"ahereas 1)oth oi these aspects are well under-
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stood for Naegleria PAM. Concomitant infections other than in the CNS have been reported in the Acanthamoeba PAM, e.g., in the prostate (15), uterus (15), skin (9), eye (15); and intestine. Often these have occurred before appearances of CNS infections, suggesting secondary hematogenous spread to the CNS. Although little clinical evidence exists to support invasion along the olfactory paths, experimental infections would suggest that occasionally this route also may be followed (20). The clinical picture of non-Naegleria infections is generally subacute or chronic, and most resemble that seen with a brain abscess or abscesses, whereas it is acute and fulminant for PAM due to Naegleria. There is no effective treatment known for Acanthamoeba infections, while amphotericin B, the antifungal agent, is highly effective against Naegleria. For non-Naegleria infections, involvement of the CNS is often diffuse and includes many midline structures, e.g., pons, medulla, caudate nucleus, and thalamus. On the other hand Naegleria invariably involves the olfactory lobe, and often confines its spread locally to temporal, frontal, and occasionally cerebellar lobes. The CSF in non-Naegleria infections usually contains a preponderance of round cells, normal or slightly low sugar, modest elevation of proteins, and amebas have not been seen or cultured from it. On the other hand, in Naegleria infections, amebas have often been seen and cultured from the CSF, the cellular reaction is predominantly neutrophilic, and the sugar is often markedly low. In conclusion: 1. PAM due to non-Naegleria organisms probably is most often due to Acanthamoeba sps.; however, a variety of free-living amebas may be involved. 2. The clinical picture of such infections is generally chronic (but occasionally may be acute), resembles that of a brain abscess(es), and more commonly occurrs in the compromised host. 3. Organs other than the brain may be involved, as infection of the CNS appears to result from hematogenous spread from a distant primary site. 4. The disease to our knowledge is invariably fatal, and medical treatment is not yet available. 5. The epidemiology is unclear, although such amebas may be found worldwide, are highly ubiquitous in Nature, are commonly found in soil and water, and even may be found in the normal upper respiratory passages of man. 6. Prevention is not employed because we don't know what to do, but natural immunity probably exists. Public health measures to control or prevent such infections may be impossible or impractical, because of the diffuseness of the problem and the variety, large numbers, and widespread natural distribution of the organisms responsible.
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REFERENCES 1. CULBERTSON, C. G., SMITH, J. AND MINNER, J.: Acanthamoeba: observations on animal pathogenicity. Science 127: 1506, 1958. 2. FOWLER, M. AND CARTER, R. F.: Acute pyogenic meningitis probably due to Acantha-
moeba sp. Brit. Med. J. 2: 740, 1965. 3. PAGE, F. C.: A revised classification of the Gymnamoebia (Protozoa Sarcodina). Zool. J. Linnean Soc. 58: 61, 1976. 4. DERRICK, E. H.: A fatal case of generalized amoebiasis due to a protozoan closely resembling, if not identical with, Iodamoeba butschlii. Trans. Roy. Soc. Trop. Med. Hyg. 42: 191, 1948. 5. KERNOHAN, J. W., MAGETH, T. B. AND SCHLOSS, G. T.: Granuloma of brain probably due to Endolimax williamsi (Iodamoeba butschlii). Arch. Path. 70: 576, 1960. 6. RINGSTED, J., JAGER, B. V., SUK, D., AND VISVESVARA, G. S.: Probable Acanthamoeba Meningoencephalitis in a Korean Child. Amer. J. Clin. Path. 66: 723, 1976. 7. JAGER, B. V. AND STAMM, W. P.: Brain abscesses caused by free-living amoeba probably of the genus Hartmannella in a patient with Hodgkin's disease. Lancet 2: 1343, 1972. 8. PATRAS, D. AND ANDUJAR, J. J.: Meningo-encephalitis due to Hartmannella (Acanthamoeba). Amer. J. Clin. Path. 46: 226, 1966. 9. GRUNDY, R. AND BLOWERS, R.: A case of primary amoebic meningoencephalitis treated with chloroquine. E. Afr. Med. J. 47: 153, 1970. 10. KENNY, M.: The micro-Kolmer complement fixation test in routine screening for soil ameba infection. Hlth. Lab. Sci. 8: 5, 1971. 11. ROBERT, V. B. AND RORKE, L. B.: Primary amebic encephalitis, probably from Acanthamoeba. Ann. Intern. Med. 79: 174, 1973. 12. NAPOLITANO, J. J., GRILLO, R. S., AND GAMBLE, H. R.: Primary Amebic Meningoencephalitis. New York State J. Med. 78: 1883, 1978. 13. HOFFMAN, E. O., GARCIA, C., LUNSETH, J., McGARRY, P., AND COOVER, J.: A case of primary amebic meningoencephalitis. Light and electron microscopy, and immunohistologic studies. Amer. J. Trop. Med. Hyg. 27: 29, 1978. 14. DUMA, R. J., HELWIG, W. B., AND MARTINEZ, A. J.: Meningoencephalitis and brain abscess due to a free-living amoeba. Ann. Intern. Med. 88: 468, 1978. 15. MARTINEZ, A. J., SOTELO-AVILA, C., GARCIA-TAMAYO, J., MORON, J. T., WILLAERT, E., AND STAMM, W. P.: Meningoencephalitis due to acanthamoeba sp. Pathogenesis and clinico-pathological study. Acta Neuropath. 37: 183, 1977. 16. BHAGWANDEEN, S. B., CARTER, R. F., NAIK, K. G., AND LEvIrn, D.: A case of hartmannellid amebic meningoencephalitis in Zambia. Amer. J. Clin. Path. 63: 483, 1975. 17. POWERS, P.: Primary amebic meningoencephalitis-New York. Morbidity and Mortality Weekly Report. 27: 343, 1978. 18. WANG, S. S. AND FELDMAN, H. A.: Isolation of Hartmannella species from human throats. New Eng. J. Med. 277: 1174, 1967. 19. CHANG, S. L., HEALY, G. R., MCCABE, L., SHUMAKER, J. B., AND SCHLUTZ, M. G.: A strain of pathogenic Naegleria isolated from a human nasal swab. HIth Lab. Sci. 12: 1, 1975. 20. MARTINEZ, A. J., MARKOWITZ, S. M., AND DUMA, R. J.: Experimental pneumonitis and encephalitis caused by Acanthamoeba in mice: pathogenesis and ultrastructural features. J. Infec. Dis. 131: 692, 1975.
DISCUSSION DR. HOOK (Charlottesville): Thank you for a most interesting paper. Do these amoebas
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produce infection in animals and it so, do vou find impressive tropism for the central nervous system? DR. DUMA (Richmond): Yes, they do. 'l ropism for the CNS certainly exists for Naegleria, as naeglerial infections seem to be linited to the CNS. One can produce infections with N. fowleri in a wide variety of inoculationi routes. With Acanthamoebas, CNS tropism appears to be less certain, although it appears that every species of Acanthamoeba (and there are many) carries the potential to produce infection. Some common animals infected by freeliving amebas are the marine animals; indeed, sizeable epidemics have occurred in mollusks, oysters, and crabs. Also, natural infections in several mammals have been obser',ved incidentally, although no systematic search for such infections has been made. DR. WITHAM (Augusta): Is it known what the incidence of infections from ponds might be, say fresh water ponds in Virginia, from both the Naegleria and non-Naegleria amoebae'? Also, does chlorine kill them in your swimming pool? DR. DUMA: No cases of PAM has-e been acquired from swimming pools in this countrY (USA), especially not chlorinated pools. In many of the European countries where heate(d pools exist, a number of cases and even tpidemics have occurred; for example, in Belgiulml and in Czechoslovakia. In Virginia where recentlv we have completed a 3 vear study of the ecologv of several lakes surrounding the city of Richmond (and I know Dr. Hook won't like to hear this, since he lives nearby) pathogenic Naegleria have been isolated on numerous occasions from one of the lakes. This lake is interesting because it serves as a coolant for a nearby electrical power plant; and a
