Opportunistic Free-Living Amebae, Part II

Clinical Microbiology Newsletter Vol. 30, No. 21 www.cmnewsletter.com $88 November 1, 2008 Opportunistic Free-Living Amebae, Part II* G.S. Visvesvara, Ph.D.1 and F.L. Schuster, Ph.D.,2 1Division of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta, GA, 2Viral and Rickettsial Disease Laboratory, California Department of Public Health, Richmond, California Abstract The free-living amebae Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri have been recognized as etiologic agents of amebic encephalitis. Although each has its own morphologic, ecologic, and epidemiologic traits, they have in common the ability to tolerate mammalian body temperature and cause infections. The amebic diseases are difficult to diagnose clinically, leading to delay in treatment, resulting in a high mortality rate. The major tests for the diagnosis of these diseases include immunostaining for amebae in brain and other tissues and conventional and real-time PCR. Balamuthia causes a subacute, though deadly, infection in either healthy or immunocompromised hosts, while Naegleria fowleri produces a fulminant infection invariably associated with exposure to freshwater. In Part II of this article, cultivation, the diseases, pathophysiology of infections, mechanisms of pathogenesis, therapy and prognosis, and, finally, prevention and control are reviewed for both of these free-living amebae. Little is known about Sappinia because there is but a single case on record. Cultivation Balamuthia mandrillaris, unlike Acanthamoeba, cannot be cultivated on bacterium-coated agar plates, and its food source in nature is not clearly known (20). It has, however, been isolated from the environment and is believed to feed on small amebae because it can feed on Acanthamoeba and Naegleria in vitro (20). It can be isolated from human or animal tissue using mammalian cell cultures, such as monkey kidney (E6), human lung fibroblasts (HLF), and human brain microvascular endothelial cells (HBMEC). In an elegant study using phase-contrast microscopy and movies, Dunnebacke (60) has shown that Balamuthia trophozoites invade cultured *Editor’s Note: Part I of this article was published in the September 15, 2008 issue of CMN (Vol. 30, No. 20). Mailing Address: Govinda S. Visvesvara, Ph.D., Division of Parasitic Diseases, Chamblee Campus, Bldg. 109, M.S.-F-36, 4770 Buford Highway NE, Atlanta, GA 30341-3724. Tel.: 770-488-4417. Fax: 770-488-4253. E-mail: [email protected] Clinical Microbiology Newsletter 30:21,2008 mammalian cells by extending pseudopodia into the cytoplasm of the cells and causing visible damage to the cells upon retracting the pseudopodia. Sometimes whole amebae enter and move around the mammalian cell and then remain quiescent for hours. Balamuthia can also be grown axenically in a complex cell-free medium containing fetal bovine serum (20). In another study, Matin et al. (61) showed that when Balamuthia was cocultured with monkey kidney fibroblast-like cells (COS-7), HBMEC, Acanthamoeba, and Escherichia coli, optimal growth occurred on HBMEC compared to COS-7 cells. Balamuthia, however, did not grow on E. coli, but when co-cultured with Acanthamoeba trophozoites, B. mandrillaris caused partial damage to Acanthamoeba. Acanthamoeba, however, encysted, and Balamuthia did not ingest the Acanthamoeba cysts. obtained at autopsy (62). CSF examination, in general, reveals lymphocytic pleocytosis with mild to severe elevation (≥1,000 mg/dl) of protein and normal or low glucose concentrations. B. mandrillaris has been identified antemortem in brain biopsy specimens from several patients; in the majority of cases, however, final diagnosis was made only at autopsy. It is difficult to differentiate B. mandrillaris from Acanthamoeba spp. in tissue sections by light microscopy because they look similar. However, they can be differentiated by immunofluorescence analysis of the tissue sections using rabbit anti-Acanthamoeba or antiB. mandrillaris sera. They can also be differentiated by transmission electron microscopy because ultrastructurally, the two amebae differ in their cyst morphology. Balamuthia can be isolated from clinical specimens provided fresh specimens are inoculated onto mammal- Diagnosis: clinical and laboratory methods B. mandrillaris, like Acanthamoeba spp., is not readily isolated from the cerebrospinal fluid (CSF). Only in one case has it been isolated from the CSF © 2008 Elsevier 0196-4399/00 (see frontmatter) 159 ian cell cultures within a day or so after biopsy or autopsy. In vitro cultures of several isolates have also been recovered from frozen brain specimens, probably because cysts, if present, may survive the freezing process while trophozoites are destroyed (1,4). Pathophysiology of Balamuthia infections Clinical manifestations of Balamuthia granulomatous amebic encephalitis (GAE) are similar to those of GAE caused by Acanthamoeba. Balamuthia GAE is chronic and develops insidiously. The incubation period may last as long as 2 years. Cutaneous lesions or ulcers may appear initially, followed by neurological symptoms as amebae invade the central nervous system (CNS). This is especially true in Peruvian patients, who develop painless skin lesions appearing as plaques a few millimeters thick and one to several centimeters wide. The lesions are commonly seen in the center of the face, but they may occur on the trunk, hands, and feet accompanied by rhinitis before CNS involvement (63). Balamuthia cases have occurred in children and elderly individuals with no known immunodeficiency, as well as in patients with HIV/AIDS and other immunodeficiencies or diabetes and those receiving prolonged corticosteroid therapy (1-5). The CNS symptoms commonly manifest initially as headache, meningismus, nausea and vomiting, low-grade fever, and lethargy. Later, other signs, such as visual disturbances, cranial nerve palsies, ataxia, seizures, and hemiparesis appear. The disease progresses to coma and eventually death from brain stem herniation and multiorgan failure, with CSF findings including elevated protein, lymphocytic pleocytosis up to 100%, and low or normal glucose concentrations. Initially, magnetic resonance imaging (MRI) may show one or more low-density lesions even when computed 160 0196-4399/00 (see frontmatter) tomography (CT) scans are unremarkable. During the course of the infection, the lesions may increase in size and number and may involve the cerebral hemispheres, cerebellum, brain stem, or thalamus. CT and MRI may indicate hemorrhage within lesions, and angiography may demonstrate occluded blood vessels corresponding to areas of infarction. The brain scans may also show “spaceoccupying lesions,” which may mimic a brain abscess, brain tumor, or intracerebral hematoma and, hence, may not specifically identify the opportunistic amebae, and some balamuthiasis patients have been erroneously diagnosed as having neurotuberculosis or neurocysticercosis. In fatal cases, the brain on gross examination appears to be edematous with evidence of uncal and tonsillar herniation. Multiple areas of meningeal softening and inflammation are seen extending into the white matter in the brainstem, cerebral hemispheres, and cerebellum. The hemorrhagic necrosis and inflammatory infiltrates consist of neutrophils, mononuclear cells, and multinucleated giant cells. Trophozoites and cysts of Balamuthia are seen extracellularly or within macrophages, which also contain lipid, and in some cases, the nuclei of the trophozoites may contain two or three nucleoli (Fig. 2C). The walls of blood vessels are surrounded and infiltrated with amebae, which may provoke vasculitis and thrombosis. In some patients, especially those with advanced HIV disease, the inflammatory reaction is sparse and granulomas are not present (1-5). Mechanisms of pathogenesis Balamuthia, like Acanthamoeba, probably invades human tissue by producing enzymes and ingesting host tissue as a food source. Recently, it has been shown that Balamuthia induces HBMEC to release a pleiotropic cyto- © 2008 Elsevier kine, interleukin 6, which is known to play a role in initiating early inflammatory response (62). Further, metalloprotease activity that may play an important role in the degradation of the extracellular matrix to produce CNS pathology has been shown in two isolates of Balamuthia (62). More recently, however, it has been shown that B. mandrillaris interacts with the host connective tissue containing extracellular matrix (ECM) proteins, such as collagen 1, fibronectin, and laminin 1 (64). According to that study, Balamuthia has a greater binding activity to laminin than to collagen or fibronectin, and scanning electron microscopy demonstrated that binding to ECM is associated with changes in the shape of the trophic amebae and production of surface projections, or food cups, reminiscent of those seen with Naegleria fowleri and Acanthamoeba spp. (64). Molecular characterization Currently, molecular techniques are being used, not only to identify these amebae, but also to understand the phylogeny and epidemiology of the genus Balamuthia (65,66). A PCR probe consisting of a primer pair specific for Balamuthia has been developed from sequence data of mitochondrial 16S rRNA genes. Using this probe, DNA of a clinical isolate obtained from the brain of a child was found to be identical to that of an ameba isolated from flowerpot soil in the child’s home (67). PCR has also been used to confirm Balamuthia GAE in a Portuguese boy (68). It has also been shown that PCR can retrospectively confirm Balamuthia infections in archived slide specimens fixed in formalin and embedded in paraffin (69). Additionally, a real-time, multiplex PCR assay specifically identifies Balamuthia DNA in human CSF and brain tissue in about 5 hours (52). Immunology Indirect immunofluorescence assay Clinical Microbiology Newsletter 30:21,2008 (IFA) and flow cytometry techniques have been used to identify Balamuthia antibodies in the sera of healthy persons, as well as patients with Balamuthia GAE (70). In a study conducted in California on patients hospitalized with encephalitis, it was found that about 3% of approximately 225 sera had anti-B. mandrillaris antibody titers of 1:128 to 1:256 in the IFA. These California patients were subsequently confirmed as being infected with B. mandrillaris by specific indirect immunofluorescence (IIF) test of biopsy sections using rabbit anti-B. mandrillaris serum and by PCR (71). Two patients who survived Balamuthia GAE infection had titers of 1:128 in the acute phase that dropped to 1:64 in convalescent sera (70). Balamuthia GAE has also been reported in a variety of animals, including gorillas, baboons, gibbons, monkeys, horses, sheep, and dogs (1,4,57-59). An animal model using severe combined immunodeficient mice has been developed to study Balamuthia GAE (71). It has been shown that in immunodeficient mice, when infected intranasally with B. mandrillaris, the amebae adhere to the nasal epithelium, migrate along the olfactory nerves, traverse the cribriform plate of the ethmoid bone, and invade the brain, similar to the invasion pathway described for N. fowleri (72). In a more recent experiment, it was shown that Balamuthia amebae migrate to the CNS after oral infection of both immunocompetent and immunodeficient mice, and Balamuthia antigen, but not organisms, has been found in mouse fecal pellets (73). Therapy and prognosis Although a majority of patients with Balamuthia GAE have died, a few patients have survived the infection because of treatment with a combination of antimicrobials. For example, three patients, a 60-year-old man, a 6-year-old girl from California, and a 70-year-old woman from New York, survived balamuthiasis after treatment with a combination of pentamidine isethionate, sulfadiazine, macrolide antibiotics (azithromycin/clarithromycin), fluconazole, and flucytosine (5-fluorocytosine) (74,75). Two of the Peruvian patients with cutaneous lesions became well after prolonged therapy with albendazole and itraconazole. One of the Peruvian patients had a large lesion on the chest wall that was Clinical Microbiology Newsletter 30:21,2008 surgically removed. Surgical excision of the lesion may have reduced the parasite load, thus helping in the recovery process (63). Currently, two patients, one a 7-year-old girl and the other a 35-year-old man, both of Hispanic ethnicity and with Balamuthia GAE, are being treated with multiple drugs as described above. The 7-year-old girl developed fever, seizures, and multiple ring-enhancing brain lesions consistent with abscess. A brain biopsy revealed focal granulomas with necrosis; chronic inflammation with histiocytes, lymphocytes, and plasma cells; and multi-nucleated giant cells with partially calcified inclusions suggestive of neurosarcoidosis, neurotuberculosis, or chronic bacterial or disseminated fungal infection. A serum sample gave a titer of 1:64 for Balamuthia by IFA; one cell in the biopsied tissue sections was positive for Balamuthia by IIF. The tissue sections were scraped from the slides, DNA was extracted, and PCR was performed and was found to be positive for Balamuthia. Currently, the patient is being treated with multiple drugs, including pentamidine, azithromycin, itraconazole, flucytosine, and sulfadiazine. The second case is that of a 35-year-old male who developed a tumor-like swelling in the brain and was initially diagnosed as having lymphoma. However, a CSF sample was positive for Balamuthia DNA and a serum sample was positive for anti-Balamuthia immunoglobulin G (IgG) antibodies with a titer of 1:256. A biopsy was performed, which was positive for Balamuthia by IIF. The patient, since leaving the hospital, is reportedly non-compliant with medication and appointments, and his current status is unknown (76). In vitro studies have shown that pentamidine and propamidine isethionates at a concentration of 1 µg/ml inhibit the growth of Balamuthia amebae by 82% and 80%, respectively, but are not amebicidal. A number of pharmaceuticals, including macrolide antibiotics, azole compounds, gramicidin, polymyxin B, trimethoprim, sulfamethoxazole, and a combination of trimethoprim-sulfamethoxazole, as well as amphotericin B, had either very little in vitro or no activity against Balamuthia isolates (70). Recently, it was shown that miltefosine lysed the amebae at a concentration of >40 μM, whereas voriconazole © 2008 Elsevier at the same concentration had no effect on Balamuthia (70). Investigations with newer formulations are urgently needed to identify drugs that would be beneficial in the treatment of this devastating infection. Epidemiology Balamuthia has been isolated from soil (67,77). It is likely that gardening or contact with dirt may play a significant role in the acquisition of Balamuthia infection by inhalation of airborne cysts or contamination of cutaneous lesions by cysts present in soil. The ameba may enter the body through a break in the skin, as probably happened to the 60-year-old man, who had been digging in his yard (74). Once it gains entry into the body, it likely spreads hematogenously to the brain. Water may serve as a source of infection, since two dogs, one from California and the other from Australia, developed balamuthiasis after swimming in stagnant ponds (78,79). A recent paper that deals with Balamuthia infection in Hispanic Americans suggests possible genetic predisposition based on as yet undetermined factors (80). Balamuthia may act as hosts for pathogenic microorganisms such as Legionella (82). Additionally, B. mandrillaris (strain CDC:V039, isolated from the mandrill baboon) supported intracellular growth of Simkania negevensis, a Chlamydia-like bacterium, but subsequently lost its ability to form cysts (82). Prevention and control Cases of balamuthiasis have occurred in immunosuppressed individuals, children, and older individuals. Presently, no clearly defined methods are available for the prevention of infection with these amebae. Naegleria fowleri N. fowleri causes primary amebic meningoencephalitis (PAM), an acute, fulminating, and hemorrhagic meningoencephalitis with abrupt onset. It occurs in previously healthy children and young adults with a history of exposure to warm freshwater about a week prior to the onset of symptoms. The first recognized case of PAM occurred in Australia in 1965 but was attributed to Acanthamoeba. The first case of N. fowleri infection in the United States was identified in Florida in 1966, and the term primary amebic meningoencephalitis was used to describe it. 0196-4399/00 (see frontmatter) 161 PAM is not a new disease; retrospective examination of archival brain tissue has revealed cases that occurred as far back as 1901 (1-6). At the present time, more than 40 species of Naegleria have been described based on the sequence of the small subunit rRNA gene (83). Life cycle N. fowleri is also referred to as an ameboflagellate because it has a transitory, pear-shaped, non-feeding, nondividing flagellate stage. It also has an ameboid trophozoite stage and a resistant cyst stage in its life cycle. The trophozoite is a slug-like ameba, feeds in vitro on gram-negative bacteria, and reproduces by binary fission. It exhibits rapid sinusoidal movement by producing anterior hemispherical bulges or lobopodia. The posterior end, or uroid, is sticky and often has several trailing filaments to which bacteria may adhere prior to being ingested into food vacuoles. The trophozoite measures 10 to 25 mm and is characterized by a single nucleus with a prominent, centrally placed nucleolus that stains densely with chromatic dyes. The cytoplasm contains numerous mitochondria, ribosomes, and food vacuoles and a contractile vacuole (Fig. 3A). The trophozoite transforms into a flagellate stage when the ionic concentration of the surrounding environment changes but eventually reverts to the trophic stage. In the laboratory, trophozoites, when suspended in distilled water, can be induced to transform into flagellates within an hour. Like the trophic ameba, the flagellate has a single nucleus with a large nucleolus and usually has two anterior flagella, but three or four flagella may also be seen occasionally. The flagellate does not have a cytostome and hence cannot feed. It ranges in length from 10 to 16 mm (Fig. 3B). Under adverse conditions when the food supply becomes scarce or its habitat dries, the trophozoite transforms into a resistant cyst that can survive environmental stress (e.g., desiccation), but is more vulnerable than the cyst of Acanthamoeba. The N. fowleri cyst is usually spherical and double walled with a thick endocyst and a closely apposed thinner ectocyst. The wall has pores flush with its surface that may not be readily seen (Fig. 3B and C) and a single nucleus with a prominent nucleolus (4,7). N. fowleri occurs worldwide and 162 0196-4399/00 (see frontmatter) Figure 3. Naegleria fowleri from culture and in human brain tissue. (A) A trophozoite showing the nucleus (N) and two vacuoles. Differential interference contrast; magnification, ×1,000. (B) An ovate cyst with a prominent nucleus. Differential interference contrast; magnification, ×1,000. (C) A flagellate with two flagella. Differential interference contrast; magnification, ×1,000. (D) Large number of trophozoites in the brain tissue of a patient. H&E; magnification, ×250. (E) Immunofluorescence pattern of trophozoites after reacting with rabbit anti-N. fowleri serum; magnification, ×500. has been isolated from freshwater, thermal discharges of power plants, heated swimming pools, hot springs, hydrotherapy and remedial pools, aquaria, and sewage, and even from the nasal passages and throats of healthy individuals. Typically, cases of PAM occur in the hot summer months when the confluence of large numbers of people engaged in swimming, diving, and water skiing in lakes, ponds inadequately chlorinated swimming pools and other warmfresh water bodies encounter the amebae (1-6). N. fowleri is thermophilic and can tolerate temperatures of up to 45°C. Therefore, these amebae proliferate during summer months when the ambient temperature is likely to be high. Factors in infection, such as the length of time immersed in water and time spent diving, are more commonly associated with children and young adults than seniors, which may account in part for the discrepancy of infection rates in younger age groups (1-6). © 2008 Elsevier Cultivation N. fowleri can be maintained in the laboratory indefinitely on non-nutrient agar plates covered with gram-negative bacteria, such as E. coli or Enterobacter aerogenes. It can also be grown on mammalian cell cultures, like E6 and HLF monolayers. The ameba feeds voraciously on the cell culture and destroys the confluent layers within 2 to 3 days. Like Acanthamoeba and Balamuthia, N. fowleri can also be grown in cell-free axenic medium, as well as in a chemically defined medium (20). The virulence of pathogenic strains diminishes with prolonged cultivation but in some cases may be restored by animal passage or growth on tissue culture cells. In the laboratory, N. fowleri trophozoites may engulf Legionella pneumophila and thus can serve as a host for the bacteria. However, N. fowleri amebae harboring Legionella or other endosymbionts have not been described (4). Primary amebic meningoencephalitis Cases of PAM generally occur durClinical Microbiology Newsletter 30:21,2008 ing the summer months. The disease may progress rapidly and cause death within a week. Rapid diagnosis and implementation of effective antimicrobial therapy is essential for patient survival. Diagnosis: clinical and laboratory methods No distinctive symptoms or clinical features differentiate PAM from pyogenic or bacterial meningitis. The CSF may have low to normal glucose, with a high protein concentration and elevated intracranial pressure. The CSF is pleocytic with a preponderance of polymorphonuclear leukocytes (PMN) early in the course of disease, but no bacteria. In situ microscopic examination of a wet mount or CSF smear may reveal the presence of actively moving amebae or, in rare instances, flagellates. Giemsa or trichrome stains of CSF smears are helpful in delineating the nuclear morphology of the amebae and thus aid in differentiating amebae from host cells (1-6). A recently developed real-time, multiplex PCR assay can identify the DNA in CSF of all three genotypes of N. fowleri known to be present in the U.S. This usually can be accomplished within 5 hours from the time the specimen arrives in the laboratory, an important factor for a quick diagnosis, since most patients are treated initially with antibacterial drugs that are ineffective against N. fowleri (51). The earliest signs and symptoms of PAM include sudden onset of bifrontal or bitemporal headaches, high fever, and nuchal rigidity, followed by nausea, vomiting, irritability, and restlessness. Later symptoms may include photophobia, lethargy, seizures, confusion, coma, diplopia, or bizarre behavior preceding death (6). Since symptoms of PAM overlap with those of bacterial meningitis, it is imperative that clinicians evaluate the patient’s history for possible freshwater exposure to arrive at the diagnosis of PAM. Pathophysiology of N. fowleri infections The olfactory bulbs of the patient are characterized by hemorrhagic necrosis and are usually surrounded by purulent exudates. The cerebral hemispheres are usually soft, swollen, edematous, and severely congested. The leptomeninges (arachnoid and pia mater) are severely congested, diffusely hyperemic, and opaque with limited purulent exudate Clinical Microbiology Newsletter 30:21,2008 within the sulci, base of the brain, brain stem, and cerebellum. The cortex also shows numerous superficial hemorrhagic areas. Most lesions are found in and around the base of the orbitofrontal and temporal lobes, base of the brain, hypothalamus, midbrain, pons, medulla oblongata, and upper portion of the spinal cord. CT scans show obliteration of the cisternae around the midbrain and the subarachnoid space over the cerebral hemispheres. Marked diffuse enhancement in these regions may be seen after administration of intravenous contrast medium (1-6). Microscopically, the cerebral hemispheres, brain stem, cerebellum, and upper portion of the spinal cord are filled with fibrino-purulent leptomeningeal exudate containing predominantly PMN and a few eosinophils, macrophages, and some lymphocytes. Large numbers of amebic trophozoites without the presence of PMN, are seen within edematous and necrotic neural tissue (Fig. 3D). Trophic amebae, ranging in size from 8 to 12 mm, can be recognized by their large, densely staining nucleoli in Virchow-Robin spaces, usually around blood vessels, with no inflammatory response. The amebae can be specifically identified as N. fowleri by histochemical techniques (IIF) using polyclonal or monoclonal antibodies (Fig. 3E). Amebic cysts are conspicuously absent. N. fowleri amebae proliferate in brain tissue, meninges, and CSF and can be cultured from samples of CSF or from brain tissue obtained at postmortem. With a few exceptions (see below), all cases of PAM reported in the literature have been fatal (1-6,84). Mechanisms of pathogenesis Amebae enter the CNS via the nasal passages and pass through the cribriform plate, penetrate into the subarachnoid space, and migrate to the brain parenchyma. The incubation period is believed to be from 2 to 15 days. N. fowleri amebae, when inoculated onto tissue culture cells, destroy the cell monolayer by causing cytopathic effects (CPE) or physical destruction. The amebae produce sucker-like appendages, or amebostomes, that “nibble” away at the tissue culture cells. Other possible factors involve the production of (i) phospholipase A and B activity, causing destruction of cell membranes; (ii) neu© 2008 Elsevier raminidase or elastase activity facilitating cell destruction; (iii) a perforin-like, pore-forming protein that lyses target cells; and (iv) a cytopathic protein that triggers apoptosis in susceptible tissue culture cells (4). The laboratory mouse can be inoculated intranasally with N. fowleri trophozoites to produce a disease that closely parallels human PAM. According to one study, mice inoculated with N. fowleri evoked an IgG response that apparently conferred protection after subsequent challenge with virulent amebae. Domesticated, as well as wild, mammals, including raccoons, muskrats, squirrels, and rabbits, develop antibodies to Naegleria amebae. Further, sera from some wild animals (raccoons, muskrats, squirrels, and bull frogs but not toads or box turtles) possessed amebicidal activity that was destroyed upon heating, implicating possible complement involvement. Close contact with soil and water makes it likely that PAM occurs in such animals also (4). Recently, PAM has been seen in the South American tapir and domestic cattle (85,86). Immunology Since most PAM patients die within a short time (5 to 10 days), there is insufficient time to mount a detectable antibody response; hence, the utility of serologic tests in the diagnosis of PAM is limited. However, a specific antibody response was documented in a California patient who recovered from PAM (84). An IgG N. fowleri-specific titer of 1:4,096 was demonstrated by IFA in the serum samples collected 7, 10, and 42 days after admission to the hospital, and the antibodies persisted even after 4 years. An immunoblot study revealed that IgM was the principal class of antibody generated by this patient, as well as by three others who contracted PAM. Additionally, sera collected from several individuals with a history of extensive swimming in freshwater lakes in the southeastern U.S., as well as in California, also revealed IgM antibodies to N. fowleri. These antibodies are particularly well developed against N. fowleri antigens of approximately 190, 66, 30, and 14 kDa. Whether these antibodies have protective activity is not clear (1). A previous study that used an agglutination test on serum specimens obtained from humans in North Carolina, Pennsylvania, and Virginia demonstrated 0196-4399/00 (see frontmatter) 163 antibody specificity for the surfaces of particular Naegleria spp., because people from these geographic areas were exposed to these antigens more extensively than people from other parts of the country. According to that study, sera from individuals residing in southeastern states had significantly greater agglutinating ability than those obtained from Pennsylvania, a northeastern state. The study showed that the agglutinating antibody is of the IgM class (87). Summarizing several published studies, (i) >80% of hospitalized patients in Tennessee had IgG and IgM antibodies with titers ranging from 1:20 to 1:640 to N. fowleri and Naegleria lovaniensis; (ii) serum specimens collected in Virginia possessed age-dependent agglutinating antibodies of the IgM class to N. fowleri; (iii) antibodies to pathogenic and nonpathogenic Naegleria ranging in titer from 1:2 to 1:120 were detected in healthy New Zealanders, and the antibody belonged mainly to IgM and IgG classes but failed to neutralize N. fowleri; (iv) mice immunized intraperitoneally with live N. fowleri were more resistant to subsequent intranasal challenge; (v) protective immunity to N. fowleri can be transferred to syngenic mice by immune serum but not by immune spleen cells; and (vi) multiple doses of N. fowleri culture supernatants were superior to multiple doses of lysate in affording protection to mice. The protective activity of the culture supernatants resided primarily in a 200,000molecular-weight fraction that is released into the medium (1-6). Molecular techniques Naegleria spp. are more or less phenotypically alike, making specific microscopic identification of N. fowleri difficult. Biochemical techniques, such as isoenzyme analysis, have been developed for the specific identification of N. fowleri amebae cultured from the CSF and brain specimens of patients, as well as from the environment (water and soil) (4). Further, monoclonal antibodies (MAb) that can specifically identify N. fowleri in the CSF have also been developed (88). Molecular techniques, such as PCR and nested-PCR assays, for the specific identification of N. fowleri in cultured amebae from patients and the environment, as well as N. fowleri DNA in the environment, have been developed (83,89-91). Sequencing of the 164 0196-4399/00 (see frontmatter) 5.8S rRNA gene and the internal transcribed spacer 1 (ITS1) and ITS2 of N. fowleri has shown that specific genotypes can be distinguished. Based on the sequencing of the ITS of the clinical isolates, it has been shown that two strains of N. fowleri, isolated from two PAM patients who were exposed to the same hot spring in California but at different times, belonged to the same type II genotype (90). A real-time multiplex PCR assay can identify N. fowleri DNA in the CSF and brain tissue samples obtained from PAM patients antemortem. This test identifies all three genotypes known to be present in the U.S. (51). A new sensitive, rapid, and discriminating technique that uses a single primer set and the DNA-intercalating dye SYT09 for real-time PCR and melting-curve analysis of the 5.8S rRNA gene flanking the ITS has been developed that distinguishes several Naegleria species in environmental samples (92). The new matrix-assisted laser desorption ionization-time of flight mass spectrometry technique mentioned earlier (93) can also specifically identify Naegleria amebae (93). Therapy and prognosis One of the few survivors of PAM, a California girl, was treated aggressively early in the course of disease with intravenous and intrathecal amphotericin B and with miconazole and oral rifampin (84). Over a 4-year follow-up, she remained healthy and free of any neurologic deficits. It is possible that amphotericin B and miconazole had a synergistic effect but that rifampin had no specific amebacidal properties. Based on in vitro testing and in vivo mouse studies, amphotericin B was reported to be more effective against Naegleria than amphotericin B methyl ester, a water-soluble form of the drug. In vitro studies of phenothiazine compounds (chlorpromazine and trifluoperazine), which can accumulate in the central nervous system, were found to have inhibitory effects on N. fowleri (4). Azithromycin, a macrolide antimicrobial, has been shown to be effective against Naegleria both in vitro and in vivo (mouse model of disease) (94). Other macrolides (erythromycin and clarithromycin) are less effective. N. fowleri was susceptible to the triazole compound voriconazole; low concentrations (≤10 μg/ml) were amebastatic, © 2008 Elsevier while concentrations ≥10 mg/ml were amebacidal (53). Prevention and control N. fowleri is a thermophilic ameba and hence proliferates in water when the ambient temperature increases above 30oC. With global warming, it is possible that cases of N. fowleri PAM may be seen in countries where it had previously not been recorded (91). Since N. fowleri is susceptible to chlorine in water (one part per million), proliferation of the amebae can be controlled by adequate chlorination of heavily used swimming pools, especially during summer months. However, it is impractical to chlorinate natural bodies of water, such as lakes, ponds, and streams, where N. fowleri may proliferate. Sunlight and the presence of organic matter in swimming pools can reduce the efficacy of chlorine. In high-risk areas, monitoring of recreational waters for N. fowleri amebae should be considered by local public health authorities and appropriate warnings should be posted, particularly during the hot summer months. Warning children not to immerse their heads in suspect waters may be judicious. In both Australia and France, where swimming pools and thermal effluents from nuclear power plants, respectively, are possible sources of infection, such monitoring of water is routine (4). If there is a single source of infection, such as a popular swimming area, a minor outbreak of PAM may occur over a period of time. Sixteen deaths due to PAM over a 3-year period were retrospectively traced to a swimming pool in Czechoslovakia with a low free chlorine concentration (4). The source of amebae was probably a grassy plot that swimmers walked across, carrying soil particles into the pool. In Arizona, two children playing in a wading pool filled from the domestic water supply developed PAM (95). N. fowleri was identified in the water by nested PCR analysis (96). This was the first time that a domestic water supply was implicated as the source of N. fowleri infection in the U.S. In Australia, domestic water carried overland in pipes warmed by the sun was also implicated as the source of infection through nasal aspiration. Drinking ameba-containing water has never been known to cause PAM. Because of a cluster of cases of PAM Clinical Microbiology Newsletter 30:21,2008 involving children in South Australia, the South Australia High Commission established an ameba-monitoring program, which routinely determined residual chlorine levels and the total coliform counts as possible predictors of “blooms” of N. fowleri. Authorities also conducted a successful campaign to educate the public on the dangers of naegleriasis in order to minimize the incidence of PAM (6). Sappinia diploidea Gelman et al. (97) reported the first and only case of amebic encephalitis caused by Sappinia diploidea in an immunocompetent, previously healthy 38-year-old male. The patient lost consciousness for about 45 minutes and developed nausea and vomiting, followed by bifrontal headache, photophobia, and blurred vision lasting 2 to 3 days. His prior history was unremarkable except for a recent sinus infection. MRI showed a solitary 2-cm mass in the posterior left temporal lobe. The excised mass on sectioning showed necrotizing hemorrhagic inflammation containing amebic organisms (Fig. 4A). The life cycle of this ameba includes trophic and cyst stages. The characteristic feature of both stages is the presence of two nuclei tightly apposed to one another. Cysts were not seen in tissue sections. S. diploidea had never previously been implicated in human or animal disease, although it had been described as a coprozoic ameba found in feces of elk, bison, and cattle. The trophozoite measures 40 to 80 mm, is ovoid or oblong, and appears to be flattened with occasional wrinkles on the surface. The cytoplasm contains a contractile vacuole and food vacuoles (Fig. 4B). The mature cyst is round and measures 15 to 30 mm (Fig. 4C). S. diploidea can be cultivated on non-nutrient agar coated with bacteria (20). The discovery of Sappinia as a potential pathogen suggests that other freeliving amebae may be identified as etiologic agents of CNS disease. 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