J. Med. Microbiol. Ð Vol. 50 12001), 205±207 # 2001 The Pathological Society of Great Britain and Ireland ISSN 0022-2615

EDITORIAL

Balamuthia mandrillaris infection

The concept that certain small free-living amoebae identi®ed as due to solely on the basis such as Acanthamoeba have the potential to cause of in the tissue sections. Isolation of B. disease in animals, including man, was suggested by mandrillaris ± initially from the brain tissue of a Culbertson in 1961 [1]. This observation was based on mandrill that died of encephalitis in the San Diego remarkable experiments conducted in the late 1950s Wild Animal Park, and subsequently from human and during the manufacture of polio vaccine at the Eli Lilly other animal brain tissue ± and the production of laboratories in Indianapolis, IN, USA [1, 2]. A few speci®c anti-B. mandrillaris serum, were instrumental years later Fowler and Carter in Australia [3] and Butt in differentiating Balamuthia from Acanthamoeba [4] in the USA described the ®rst human cases of encephalitis [10, 11]. amoebic meningo-encephalitis caused by . Since then several hundred cases of central nervous system 1CNS) disease caused by Acanthamoe- Protozoology, cultural characteristics, and ba spp., N. fowleri and have epidemiology been reported worldwide [5±7]. Several cases in animals other than man have also been reported B. mandrillaris, like Acanthamoeba, has a vegetative [8, 9]. Infection is almost invariably fatal. The CNS trophic stage and a dormant stage in its life cycle. disease produced by N. fowleri is called primary The trophozoites measure 15±60 ìm in diameter and amoebic meningo-encephalitis and occurs principally in are characterised by a round nucleus with a large, immunocompetent children and young adults with a spherical, densely staining nucleolus. Binucleate forms history of contact with fresh water; infection is are occasionally seen. In some cases the nucleus may fulminating and nearly always fatal. The infection have more than one nucleolus and this feature distin- caused by Acanthamoeba spp. or B. mandrillaris is guishes B. mandrillaris amoebae from Acanthamoeba, called granulomatous amoebic encephalitis. Acantha- especially in tissue sections [7, 10]. During the early moeba encephalitis occurs principally in immunosup- stages of mitosis the nucleolus and nuclear membrane pressed individuals, chronic alcoholics and debilitated both remain intact, but disappear as mitosis progresses. persons; B. mandrillaris causes encephalitis in the very The nucleus is surrounded by abundant cytoplasm young or very old and in immunosuppressed indivi- containing empty vacuoles, numerous mitochondria, duals. Acanthamoeba spp. also cause a non-fatal, but ribosomes and endoplasmic reticulum. The trophozoites vision-threatening keratitis of the human eye [5±7]. form broad pseudopodia and move slowly. Occasionally, The true incidence of amoebic encephalitis is not the amoebae produce ®nger-like pseudopodia and exactly known. As of 1 Aug. 2000, .190 cases of exhibit spider-like walking movement across the ¯oor meningo-encephalitis due to N. fowleri have been of the tissue culture cells on which they are feeding [11]. reported worldwide, 95 in the USA alone. More than The cysts of B. mandrillaris are usually spherical and 120 cases of granulomatous encephalitis due to measure 6±30 ìm in diameter, with a mean of 15 ìm. Acanthamoeba spp. 184 in the USA, including at least They are usually uninucleate and possess a layer of 50 patients with HIV) and .3000 cases of Acantha- refractile granules beneath the inner cyst wall. Under the moeba keratitis have been reported. There have been optical they resemble Acanthamoeba cysts .85 additional reports of encephalitis due to B. with an outer wrinkled wall 1the ectocyst) and an inner mandrillaris; 40 of these occurred in the USA, and thin wall 1the endocyst), but ultrastructurally they are include .10 patients with HIV. The actual number is tripartite with an additional thick, amorphous, ®brillar probably higher, as the diagnosis is usually made at middle layer, the mesocyst [11]. autopsy, which is seldom done in many countries [5]. N. fowleri and Acanthamoeba spp. are widely dis- Until 1989, the identi®cation of the causative agent of tributed in fresh water, soil and dust throughout the granulomatous amoebic encephalitis, in the absence of world, thus providing a potential source of infection for culture isolation or immunohistological assay, was man and other animals [5±7, 12]. The environmental based on the presence of amoebic cysts in tissue niche of B. mandrillaris is not known, but it is believed sections, as N. fowleri does not produce cysts in tissue to occupy the same locations as N. fowleri and whereas Acanthamoeba does. Hence many cases were Acanthamoeba spp. 206 EDITORIAL Unlike Acanthamoeba spp. and N. fowleri, B. man- that may be helpful in the diagnosis of B. mandrillaris drillaris does not grow on non-nutrient agar plates infection has been developed [25]. coated with gram-negative [11]. It is probably for this reason that B. mandrillaris has not been Cerebrospinal ¯uid 1CSF) obtained by lumbar puncture isolated from nature, as free-living amoebae are usually may show lymphocytic pleocytosis, normal or slightly isolated by inoculating environmental samples on low glucose level, and mild elevation of , bacteria-covered agar plates. B. mandrillaris grows usually without amoebic trophozoites or cysts. The well at 35±378C on monkey kidney cells or human CSF resembles that of aseptic meningitis in most cases. lung ®broblasts, as well as in a complex chemical medium containing fetal bovine serum [13], which is Neuroimaging by computed tomography and magnetic helpful in the screening of isolates for drug suscept- resonance imaging of the head are helpful in the ibility and potential treatments [13, 14]. diagnosis [5, 23, 24]. Intravenous contrast material may reveal ring-enhancing masses suggestive of a brain Experimental animals such as out-bred mice have been abscess or a brain tumour [5, 23]. Chest X-ray may infected by intranasal instillation of culture-grown B. show focal areas of consolidation in the pulmonary mandrillaris, resulting in disease similar to that seen in tissue. man and death of the animals [10, 11]. A congenitally immunode®cient mouse model has also been described, con®rming the `opportunist' characteristics of B. Pathogenesis and pathological features mandrillaris infection [15]. B. mandrillaris produces multifocal, subacute or chronic granulomatous encephalitis with trophozoites and cysts within the central nervous system, as well as Clinical features and diagnosis cutaneous nodules, ulcerations and pneumonitis. Neu- ropathologically, infection is characterised by brain Clinically, encephalitis due to B. mandrillaris resem- oedema and a subacute necrotising haemorrhagic bles that caused by Acanthamoeba, namely the pres- encephalitis. A modest lymphocytic in®ltrate is often ence of a single or multiple space-occupying les- present, depending on the immunological status of the ions. Neurologically, hemiparesis, aphasia and seizures host. This is usually composed of CD4 and CD8 T may appear early. Personality and mental status cells and B lymphocytes, together with a few plasma abnormalities may be present, associated with head- cells, macrophages and multinucleate giant cells. ache, stiff neckand cranial nerve palsies mainly Amoebic trophozoites and cysts are usually present affecting the third and sixth cranial nerves. Cerebellar within perivascular spaces and within the necrotic CNS ataxia, diplopia and low grade fever have been reported parenchyma. Focal chronic leptomeningitis may be in some cases. These symptoms closely mimic ± and seen in the areas near the parenchymal lesions. are often mistaken for ± a bacterial or viral Arteritis with trophozoites and cysts may be seen in encephalitis, leptomeningitis and tuberculous meningi- the same areas. Pneumonitis, and ulcerative dermatitis tis. Raised intracranial pressure can lead to papilloe- with the presence of trophozoites and cysts have also dema, coma, herniation of cerebellar tonsils and death. been described [5]. Invasion and penetration into the The direct cause of death is usually acute broncho- CNS probably arises by haematogenous spread from a pneumonia, hepatic or renal failure, septicaemia, and primary focus of infection in the lower respiratory tract brain oedema leading to uncal and cerebellar tonsillar or skin [5]. The ulcerated skin lesions may serve as a herniation [5, 16±23]. portal of entry of the or may represent `terminal' haematogenous dissemination of the infec- The diagnosis is made when the amoebic trophozoites tion. and cysts are identi®ed in tissues or the agent is isolated in culture. Brain and skin biopsies are useful B. mandrillaris can infect healthy and immunosup- for culture and for histological examination with pressed hosts of both sexes, and there may be no haematoxylin and eosin or special stains like Gomori's history of swimming or exposure to contaminated methenamine silver and periodic acid-Schiff. However, water. Encephalitis due to B. mandrillaris usually runs immunohistological techniques, e.g., indirect immuno- a long protracted and insidious clinical course with an ¯uorescence assay, or electron microscopy are neces- unknown incubation period, that is de®nitely longer sary to identify B. mandrillaris in tissue sections [5± than 10 days and may last up to several months. 11, 16±24]. Molecular biological methods such as the PCR or oligonucleotide probes are not available at the present time. Biopsy or autopsy tissue ®xed in 10% Treatment, control and prognosis buffered formalin or in Karnovsky's ®xative can be embedded in plastic for electron microscopy to There is presently no known effective treatment for demonstrate the characteristic ultrastructural features encephalitis due to B. mandrillaris and the prognosis is of trophozoites and cysts [5]. A serum test very poor. Experimental studies indicate that the EDITORIAL 207 amoeba is sensitive to pentamidine isethionate, azi- mandrillaris 1leptomyxid ameba) in a horse. J Vet Diagn thromycin and clarithromycin [13, 14]. One patient Invest 1998; 10: 378±381. 10. Visvesvara GS, MartõÂnez AJ, Schuster FL et al. Leptomyxid with biopsy-con®rmed B. mandrillaris encephalitis ameba, a new agent of amebic meningoencephalitis in humans survived after treatment with the following regimen: and animals. J Clin Microbiol 1990; 28: 2750±2756. clarithromycin 500 mg three times a day; ¯uconazole 11. Visvesvara GS, Schuster FL, Martinez AJ. Balamuthia mandrillaris, N. G, N. Sp., agent of amebic meningoencepha- 400 mg daily; sulphadiazine 1.58 g every 6 h, and 5- litis in humans and other animals. J Eukaryot Microbiol 1993; ¯uorocytosine 1.5 g every 6 h. However, the patient 40: 504±514. suffered severe neurological de®cits 1Dr T. Deetz, 12. John DT. Opportunistically pathogenic free-living amebae. In: Kreier JP, Baker JR 1eds) Parasitic , vol III, 2nd edn. Santa Cruz Medical Clinic, CA, USA, personal San Diego, Academic Press. 1999: 143±246. communication to G.S.V.). 13. Schuster FL, Visvesvara GS. Axenic growth and drug sensitivity studies of Balamuthia mandrillaris, an agent of We thankMary Bartlett and Rizwan Aslam for editorial assistance. amebic meningoencephalitis in human and other animals. J Clin Microbiol 1996; 34: 385±388. Â Ã 14. Schuster FL, Visvesvara GS. Ef®cacy of novel antimicrobials A. JULIO MARTINEZ and GOVINDA S. VISVESVARA against clinical isolates of opportunistic amebas. J Eukaryot University of Pittsburgh Medical Center 1UPMC), Microbiol 1998; 45: 612±618. Pathology Department, Neuropathology Division, 15. Janitschke K, MartõÂnez AJ, Visvesvara GS, Schuster F. Animal Presbyterian Hospital, model of Balamuthia mandrillaris CNS infection: contrast and Pittsburgh, PA 15213, USA comparison in immunode®cient and immunocompetent mice: a 1e-mail: [email protected]), murine model of ``granulomatous'' amebic encephalitis. J and ÃDivision of Parasitic Diseases, Neuropathol Exp Neurol 1996; 55: 815±821. National Center for Infectious Diseases, 16. Anzil AP, Rao C, WrzolekMA, Visvesvara GS, Sher JH, Centers for Disease Control and Prevention, Kozlowski PB. Amebic meningoencephalitis in a patient with AIDS caused by a newly recognized opportunistic , Atlanta, GA 30333, USA Leptomyxid ameba. Arch Pathol Lab Med 1991; 115: 21±25. 1e-mail: [email protected]) 17. Taratuto AL, Monges J, Acefe JC, Meli F, Paredes A, MartõÂnez Corresponding author: Dr A. J. MartõÂnez AJ. Leptomyxid ameba encephalitis: report of the ®rst case in Argentina. Trans R Soc Trop Med Hyg 1991; 85: 77. 18. Gordon SM, Steinberg JP, DuPuis M, Kozarsky P, Nickerson JF, Visvesvara GS. Cultural isolation of Acanthamoeba sp. and References Leptomyxid amebas from patients with amebic meningoence- phalitis, including two patients with AIDS. Clin Infect Dis 1. Culbertson CG. Pathogenic Acanthamoeba 1Hartmannella). Am 1992; 15: 1024±1030. J Clin Pathol 1961; 35: 195±202. 19. Griesemer DA, Barton LL, Reese CM et al. Amebic 2. Culbertson CG, Smith JW, Minner JR. Acanthamoeba: meningoencephalitis caused by Balamuthia mandrillaris. Pe- observations on animal pathogenicity. Science 1958; 127: 1506. diatr Neurol 1994; 10: 249±254. 3. Fowler M, Carter RF. Acute pyogenic meningitis probably due 20. Reed RP, Cooke-Yarborough CM, Jaquiery AL et al. Fatal to Acanthamoeba sp: preliminary report. BMJ 1965; 2: 740± granulomatous amoebic encephalitis caused by Balamuthia 742. mandrillaris. Med J Aust 1997; 167: 82±84. 4. Butt CG. Primary amebic meningoencephalitis. N Engl J Med 21. Denney CF, Iragui VJ, Uber-ZakLD et al. Amebic 1966; 274: 1473±1476. meningoencephalitis caused by Balamuthia mandrillaris: case 5. MartõÂnez AJ, Visvesvara GS. Free-living, amphizoic, and report and review. Clin Infect Dis 1997; 25: 1354±1358. opportunistic amebas. Brain Pathol 1997; 7: 583±598. 22. Recavarren-Arce S, Velarde C, Gotuzzo E, Cabrera J. Amoebic 6. MartõÂnez AJ, Visvesvara GS, Chandler FW. Free-living amebic angeitic lesions of the central nervous system in Balamuthia infections. In: Connor DH, Chandler FC, Schwartz DA, Manz mandrillaris . Hum Pathol 1999; 30: 269±273. HJ, LackEE 1eds) Pathology of infectious diseases, vol II. 23. LowichikA, Rollins N, Delgado R, Visvesvara GS, Burns DK. Stamford, CT, Appleton & Lange. 1997: 1163±1176. Leptomyxid ameba meningoencephalitis mimicking brain stem 7. Visvesvara GS, Stehr-Green JK. Epidemiology of free-living glioma. Am J Neuroradiol 1995; 16 14 Suppl): 926±929. ameba infections. J Protozool 1990; 37: 25S±33S. 24. Katz JD, Ropper AH, Adelman L, Worthington M, Wade P. A 8. Rideout BA, Gardiner CH, Stalis IH, Zuba JR, Had®eld T, case of Balamuthia mandrillaris meningoencephalitis. Arch Visvesvara GS. Fatal infections with Balamuthia mandrillaris Neurol 2000; 57: 1210±1212. 1a free-living amoeba) in gorillas and other Old World 25. Huang ZH, Ferrante A, Carter RF. Serum to primates. Vet Pathol 1997; 34: 15±22. Balamuthia mandrillaris, a free-living amoeba recently demon- 9. Kinde H, Visvesvara GS, Barr BC, Nordhausen RW, Chiu strated to cause granulomatous amebic encephalitis. J Infect PHW. Amebic meningoencephalitis caused by Balamuthia Dis 1999; 179: 1305±1308.