Naegleri fowleri

Naegleria fowleri is the causal agent of primary amoebic meningoencephalitis (PAME).

Naegleria spp. are amoeboflagellates found in water and soil. Although, some 30 species of Naegleria have been recognized based upon sequencing data (De Jonckheere, 2004). N.

fowleri is the only one that has been isolated from cases of primary amoebic meningoencephalitis. Other species (Naegleria italica, Naegleria philippinensis, Naegleria australiensis) may be pathogenic in the mouse model of PAME, but have not been identified from any human cases. Because, it grows best at somewhat elevated temperatures, the amoeba has been isolated from warm-water bodies including ponds and lakes, hot springs, and thermally polluted streams and rivers. N. fowleri is thermotolerant, being able to survive temperatures up to 45℃, preadapting the species to mammalian body temperature. Indeed, incubation at 45℃ is routinely used to isolate the amoeba from water samples, while suppressing growth of other amoebae in the samples (De Jonckheere and van de Voorde, 1977b). But thermotolerance is not the sole factor determining pathogencity of Naegleria spp.

Naegleria Lovaniensis is a thermotolerant species, but is non-pathogenic in the mouse model for PAME (De Jonckheere and van de Voorde, 1977a; Stevens et al., 1980).

PAME is a fulminating disease, developing within several days following exposure to the water source, and causing death within 1~2 weeks after hospitalization. The ability of N.

fowleri to produce such a rapidly fatal infection has encouraged search for virulence properties of the amoeba that might account for its destructiveness. Among candidates that might serve as virulence factor are the release of the enzymes phospholipase (Cursons et al., 1978), or neuraminidase (Eisen and Franson, 1987), the creation of pores in target cell membranes that may have a lytic effect (Young and Lowery, 1989) and aggressive phagocytotic activity (Brown, 1979). The amoeboid stage forms food cups that are capable of pinching off bits of target cell cytoplasm (Brown, 1979; John et al., 1985).

(A) Life cycle, morphology, growth and culture

There are both trophozoite and cyst stages in the life cycle, with the stage primarily depending on environmental conditions. Trophozoites can be found in water or moist soil and can be maintained in tissue culture or other artificial media.

The trophozoites can occur in two forms, amoeboid and flagellate. Mortility can be observed in hanging-drop preparations from cultures of cerebrospinal fluid (CSF); the amoeboid form is elongate with a broad anterior end and tapered posterior end. The size ranges from 7 to 35 ㎛. The diameter of the rounded forms is usually 15 ㎛. There is a large, central karyosome and no peripheral nuclear chromatin. The cytoplasm is somewhat granular and contains vacuoles. The amoeboid form organisms change to the transient, pear-shaped flagellate form when they are transferred from culture or teased from tissue into water and

maintained a temperature of 27℃ to 37℃. The change may occur very quickly or may take as long as 20 h. The flagellate form has two flagella at the brad end. Mortility is typical, with either spinning or jerky movements. These flagellate forms do not divide, but when the flagella are lost, the amoeboid forms resume reproduction.

Cysts form nature and from agar cultures look the same and have a single nucleus almost identical to that seen in the trophozoite. They are generally round, measuring from 7 to 15 ㎛, and there is a thick double wall.

In nature and in the laboratory, the amoeba feeds actively on bacteria. Isolations of N.

fowleri from environmental water and soil samples are accomplished by growth on non-nutrient agar plates coated with E. coli at 45℃ (Lares-Villa et al., 1993). Other bacteria, such as Enterobacter aerogenes or Klebsiella pneumoniae, may also be used. Once established in vitro, the amoebae can also grown in an axenic medium following elimination of bacteria by antibiotic treatment of the culture (Schuster, 2002).

(B) Pathogenicity

The pathogenesis of PAME is poorly understood. Both pathogenic and nonpathogenic species of Naegleria have been isolated from the environment, but the determinants of virulence and pathogenicity are unknown. As with many pathogenic organisms, prolonged growth of N. fowleri in axenic culture in vitro results in attenuation of virulence, while serial passage through mice restores and maintains virulence (Wong et al., 1977; Whiteman and Marciano-Cabral, 1987).

Adherence of pathogens to host cells is a critical initial step in the infection process.

The ability of trophozoites to attach to the nasal mucosal, an increased rate of locomotion, and a chemotactic response to nerve cell components may be important in disease progression (Cline et al., 1986; Brinkley and Marciano-Cabral, 1992; Han et al., 2004).

A variety of in vitro cell culture systems have been used to study the infection of N.

fowleri with mammalian cells. N. fowleri trophozoites have been shown to destroy nerve cells, as well as other cell types, by trogocytosis using food-cup structure on their surface (Brown, 1979; Marciano-Cabral et al., 1982; Marciano-Cabral and Fulford, 1986) and by the release of cytolytic molecules (Lowery and McLaughlin, 1984, 1985; Marciano-Cabral and Fulford, 1986; Leippe and Herbst, 2004). The mode that is applied to destroy target cells in vitro, however, is dependent on the amoeba strain. For example, weakly pathogenic strains destroy nerve cells by ingestion using the food-cup structure, while highly pathogenic strains lyse nerve cells on contact and subsequently ingest the cell debris that is generated (Marciano-Cabral et al., 1982; Marciano-Cabral, 1988).

(C) Primary amoebic meningoencephalitis

Once entering into the nostrils of swimmers, others engaging in water sports, and the nasal cavity by inhalation or aspiration of dust, water, or aerosols containing the trophozoites or cysts. N. fowleri penetrates the mucosal epithelial layer and migrates along the olfactory nerve tracts, crossing the cribriform plate, to the brain. The cribriform plate in children is more porous than in adults, another possible reason for the higher incidence of PAME in

young persons. Because of their proximity to the point of entrance of amoebae into the central nervous system (CNS), the frontal and olfactory lobes of the brain are the initial targets of amoebic destruction. Other areas affected are the base of the brain, the brainstem, and the cerebellum (Martinez, 1985). Amoebae are found in large numbers in the perivascular regions in brain tissue. A purulent exudates containing trophic amoebae can be found in the subarachnoid space of the meninges. Involvement of the meninges is the basis for the distinction made between PAME and Acanthamoeba and Balamuthia enceephalitides, which are typically granulomatous amoebic encephalitides. PAME can resemble acute purulent bacterial meningitis, and these conditions may be difficult to differentiate, particularly in the early stages. The CSF may have a predominantly polymorphonuclear leukocytosis, increased protein concentrations, and decreased glucose concentration like those seen with bacterial meningitis. Unfortunately, if the CSF Gram stain is interpreted incorrectly, the resulting antibacterial therapy has no impact on the amoebae and patient will usually die within several days.

Extensive tissue damage occurs along the path of amoebic invasion; nasopharyngeal mucosa shows ulceration, and the olfactory nerves are inflamed and necrotic. Hemorrhagic necrosis is concentrated in the region of the olfactory bulbs and the base of the brain.

Organisms can be found in the meninges, perivascular spaces, and sanguinopurulent exudates.

(D) Symptoms

Primary amoebic meningoncephalitis caused by N. fowleri is an acute, supperative infection of the brain and meninges. With extremely rare exceptions, the disease is rapidly fatal in humans. The period between contact with the organism and onset of clinical symptoms such as headache, fever, and rhinitis may vary from 2 to 3 days to as long as 7 to 15 days. Early symptoms include vague upper respiratory distress, lethargy, headache, and occasionally olfactory problems. The acute phase includes sore throat, stuffy blocked or discharging nose, and severe headache. Progressive symptoms include pyrexia, vomiting, and stiffness of the neck. Mental confusion and coma usually occur approximately 3 to 5 days prior to death. The cause of death is usually cardiorespiratory arrest and pulmonary edema.

(E) Diagnosis

Rapid diagnosis is by microscopic examination, preferably phase-contrast, of freshly drawn CSF, to visualize motile amoebae. Suspending amoebae in 1 ml of distilled water can further confirm the identity of the amoebae as Naegleria, by watching for development of actively swimming flagellates (Visvesvara, 1999). Further corroboration can be obtained by isolation of the amoeba from CNS or macerated brain tissue on a non-nutrient agar plate that has been spread with a lawn of E. coli, and incubated overnight at 37℃. The amoebae will grow out in large numbers, feeding on the bacteria. Because this is an acute disease, early

diagnosis is essential in order that appropriate antimicrobial therapy may be initiated before the amoebae do extensive damage. Diagnosis may be delayed when amoebae in CSF are mistaken for leukocyte being sluggish in motion, while N. fowleri move relatively swiftly with a distinctive ectoplasmic pseudopod.

(F) Treatment

Many antiparasitic and antimicrobial drugs have been screened for in vitro and in vivo activity against N. fowleri. Naegleria spp. are highly sensitive to the antifungal drug amphotericin B, and it has been used in virtually all cases as the core antimicrobial where recovery occurred. Minimum amoebacidal concentrations of amphotericin B were determined to be 0.02∼0.078 ㎍/ml for three different clinical isolates of N. fowleri tested in vitro (Duma et al., 1971). Ultrastructural examination of amoebae treated with amphotericin B revealed membrane distortions, including the nuclear envelope, rough and smooth endoplasmic reticula, and plasma membrane blebbing (Schuster and Rechthand, 1975).

Although N. fowleri is very sensitive to amphotericin B in vitro, only a few patients have recovered after receiving intravenous injections and intrathecal of this drug alone or in combination with miconazole (Schuster and Visvesvara, 2004).

Naegleria infections have also been treated successfully with amphotericin B, rifampin, and chloramphenicol; amphotericin B, oral rifampin, and oral ketoconazole; and amphotericin B alone (Abramowicz, 2004; Schuster and Visvesvara, 2004).

The variables in determining the survival of PAME cases are how early the diagnosis is made and treatment initiated, the infectious dose of amoebae, the virulence of the infecting strain and the health of the patient. It is of interest to note that the N. fowleri strain isolated from this patient appeared to be less virulent than other clinical isolates when tested for cytopathogenicity in monkey cell cultures (John and John, 1989).

The macrolide antibiotic azithromycin is effective against Naegleria in vitro and in the mouse model, but it is reported to penetrate poorly into the CSF (Schuster et al., 2001;

Goswick and Brenner, 2003a). Other antimicrobials that have been tested, mostly in vitro, are clotrimazole, itraconazole, fluconazole, and ketoconazole, with varying degrees of efficacy. Differences in reported drug sensitivity are due to the use of different N. fowleri strains in different laboratories, which show variation to drugs. Amphotericin B, however, remains the drug of choice for PAME treatment.

(G) Immunity

N. fowleri are among the protozoa that humans are exposed to in the course of their lives, either through direct contact with water or soil, or by wind-blown cysts that might lodge in the nasal mucosa. In a study of selected groups in Czechoslovakia (students, psychiatric patients), referred to earlier, percentages of individuals giving positive antibody responses for N. fowleri ranged from 1 to 4% (Cerva, 1989). Much higher percentages of positive responses were found in serum samples of hosptitalised patients in the United States in which antibodies of the IgG and IgM classes were detected to N. fowleri and the

non-pathogenic N. lovaniensis. Titters ranged from 1:20 to 1:640. IgG, but not IgM antibodies were detected in sera from newborn infants, supporting evidence for cross-placental transfer (Dubray et al., 1987). Antibodies against Naegleria were found in a variety of wild mammals including raccoons, some rodents and opposums, mirroring the presence of Naegleria antibodies in human population (Kollars and Wilhelm, 1996).

Sera obtained from wild animals showed lytic activity attributable to complement, since heat treatment destroyed amoebacidal activity (John and Smith, 1997). Of various species tested, amoebacidal activity was strongest in sera from raccoon, muskrat and bullfrog, all species closely associated with aquatic environments. Given the close association of wild animals with water and soil, it is not surprising that protective immune agents should be encountered.

Immunofluorescence staining for Naegleria antibody in PAME patients is not very effective as a diagnostic tool. The reason, of course, is that there is insufficient time for a humoral antibody reaction to occur. Differences in surface antigens exist among Naegleria spp. N. fowleri and N. lovaniensis share similar antigens (Marciano-Cabral et al., 1987).

The humoral immune response to N. fowleri has been studied in experimental animals and humans. Serum samples from healthy individuals from the United States (Reill et al., 1983a; Marciano-Cabral et al., 1987), New Zealand (Cursons et al., 1977, 1980a), and the Czech Republic (Cerva, 1989) have been examined for antibodies to N. fowleri. Although the antibody titers recorded have differed from study to study, almost all human sera from healthy individuals have been found to be positive for N. fowleri, indicating that exposure to the amoeba is common. Naegleria spp. Antibodies to N. fowleri have also been detected in

101 of 115 randomly obtained serum samples from hospitalized patients in the United States (Dubray et al., 1987). Using immunoblot analysis, the examined serum samples from army recruits with acute respiratory disease for antibodies to free-living amoebae (Powell et al., 1994).

A number of studies have been conducted to determine the role of antibody by idiotype in host resistance to infection with Naegleria. It survey to detect IgA antibodies in the serum and saliva of individuals living in Mexico in areas where Naegleria is endemic as compared with those where it is nonendemic (Rivera et al., 2001). The serum obtained before death from a patient with PAME, which showed no increase in specific antibody titers by indirect immunofluorescent antibody assay, revealed very low levels of IgA upon radial-immunodiffusion testing for quantitation of serum IgG, IgA and IgM (Cursons et al., 1979).

In contrast, the serum obtained before death from a patient with PAME and noted that the levels of serum immunoglobulins, including those of IgA, were within normal limits (Cain et al., 1979). Using immunofluorescence testing, it was reported an antibody titer of 1:4096 after 42 days of infection in a patient who survived PAME following treatment by intravenous and intraventricular administration of amphotericin B (Seidel et al., 1982).

Circulating IgG antibodies in serum were demonstrated by ELISA from day 7 after infection in ICR mice infected with N. fowleri (Park et al., 1987b) and from day 14 after infection in ICR mice infected with N. jadini, but no anti-N.gruberi antibody was detected (Lee et al., 1985).