**3. Pathogenesis**

308 Non-Flavivirus Encephalitis

containing 4, 11, and 5 species, respectively (De Jonckheere, 1987). Recently, reclassification was made to group various species into 15 genotypes (T1 to T15) based on 18S rRNA gene sequences, which can distinguish different genotypes showing a variation of as low as 5% (Gast*, et al.*, 1996, Schuster & Visvesvara, 2004). The genotypes T1, T4, T10, and T12 generally cause encephalitis, and *A. castellanii* and *A. polyphaga* that belong to T4 genotype are most frequently implicated as causes of GAE (Garate*, et al.*, 2006). The current trend is to

Epidemiologically, no clear disease associations have been found with respect to race, gender, and geographical location, except that the disease has not been reported in Africa, an observation attributed mostly to lack of familiarity and diagnostic tools (Tan*, et al.*, 1993, Barker*, et al.*, 1995, Marciano-Cabral & Cabral, 2003). However, serological evidence suggests that Hispanics are 14.5 times less likely to carry *Acanthamoeba*-reactive antibodies than individuals of other ethnicities (Chappell*, et al.*, 2001, Khan, 2006). A study undertaken to measure the seroprevalence of *Acanthamoeba* in different ethnic groups indicated that up to 90 % of healthy humans can carry *Acanthamoeba* antibodies. Seropositivity occurred in the order of Caucasians, followed by Hispanics and African Americans (Chappell*, et al.*, 2001). But it is unknown whether the occurrence of amoebic encephalitis follows a similar pattern

The amoeba has a two-stage life-cycle: trophozoites (infective and invasive) and cysts (dormant). The life cycle can be completed in either the environment or infected hosts (Chagla & Griffiths, 1974, Marciano-Cabral & Cabral, 2003). Under unfavorable conditions, such as extremes of pH and temperature, trophozoites become cysts that are highly resistant to commonly used disinfectants containing chlorine and the cysts can survive environmental temperatures even upto 80o C (De Jonckheere & van de Voorde, 1976, Khunkitti*, et al.*, 1998, Storey*, et al.*, 2004). *Balamuthia* spp. (e.g., *B. mandrillaris*) can cause GAE in a wide range of species such as horses, baboons, sheeps, dogs, and humans (Martinez & Visvesvara, 2001), but development of clinical disease takes months to years. Likewise, *Acanthamoeba* infections are also reported in humans including domestic and nondomestic species such as dogs, monkeys, kangaroos and buffaloes (Schuster & Visvesvara, 2004). Found ubiquitously, Acanthamoebae have been isolated from a variety of sources such as soil; drinking, natural and sea water; hospitals, eye wash stations, and dental irrigation systems; swimming pools; and heating and cooling ducts (Jahnes & Fullmer, 1957, Kingston & Warhurst, 1969, Casemore, 1977, De Jonckheere, 1991, Barbeau & Buhler, 2001, Marciano-Cabral & Cabral, 2003, da Rocha-Azevedo*, et al.*, 2009) and the amoebae generally feed on bacteria, algae, and yeast (Bowers, 1977, Bowers & Olszewski, 1983, Marciano-

Generally, GAE is regarded as a disease of immunocompromised individuals. HIV patients, individuals undergoing immunosuppressive and steroid therapies, and those who have received organ or stem cell transplants are at great risk of developing the disease (Marciano-Cabral*, et al.*, 2000, Seijo Martinez*, et al.*, 2000, Marciano-Cabral & Cabral, 2003, Schuster & Visvesvara, 2004, Khan, 2006, da Rocha-Azevedo*, et al.*, 2009). Other predisposing factors include malignancies and debilitated conditions such as diabetes, chronic alcoholism and

classify Acanthamoebae based on genotype rather than morphology.

**2.1 Geographical distribution** 

in the general population.

**2.2 Host distribution and susceptibility** 

Cabral & Cabral, 2003, da Rocha-Azevedo*, et al.*, 2009).

The amoebae can gain entry into the CNS through two routes: migration via the olfactory neuroepithelium and/or blood (Fig. 1). *Naegleria* spp. tend to follow the former route. After penetrating the nasal mucosa, the amoebae pass through the cribriform plate and travel along the nerve fibers to the olfactory bulb in the cerebrum (Khan, 2007, Elsheikha & Khan). Alternatively, the amoebae that enter through nasal exposure go to the lungs, enter the blood stream, and reach the CNS possibly by disrupting the blood brain barrier (BBB). The hematogenous route also is a choice for amoeba that enter through the skin (Khan, 2007). However, the mechanism by which amoebae actually enter the CNS is not clearly elucidated. Although it is postulated that they enter through the cerebral capillary endothelium or choroid plexus, the former being the more widely accepted mechanism (Khan, 2003, Marciano-Cabral & Cabral, 2003, Khan, 2005b, Khan, 2006, da Rocha-Azevedo*,* 

Autoimmunity in the Mediation of Granulomatous Amoebic Encephalitis: Implications for Therapy 311

maintained (Khan, 2003, Khan, 2006, Khan, 2007). Disruption of the BBB is thought to be mediated by contact-dependent or contact-independent mechanisms (Khan, 2003, Khan, 2006, Khan, 2007). Contact-dependent mechanisms require attachment of the amoeba to the brain's microvascular endothelial cells through amoebic mannose-binding protein (mannose-BP) causing apoptosis of the endothelial cells, a phenomenon that depends on the phosphatidylinositol 3-kinase signaling pathway (Sissons*, et al.*, 2005, Khan, 2006, Khan, 2007). In contrast, contact-independent mechanisms involve extracellular proteases secreted by *Acanthamoeba*, particularly serine proteases, and these enzymes destroy extracellular matrix proteins comprised of collagen (type I, III, and IV), elastin, and fibronectin (Khan*, et al.*, 2000, Sissons*, et al.*, 2005, Khan, 2007). Alternatively, infected immune cells, most importantly macrophages, may simply act as Trojan horses to carry the amoebae to the CNS

The pathogenicity of *Acanthamoebae* varies by species, depending on their inherent potential to tolerate temperatures, attachment to cellular surfaces, and induction of cytolysis (Marciano-Cabral & Cabral, 2003, Khan, 2006). The principal virulent factors are mannose-BP, Nicotinamide adenine dinucleotide (NADH)-dehydrogenase, GDP-mannose pyrophosphorylase and proteasomal ATPase (Marciano-Cabral & Cabral, 2003, Han*, et al.*, 2006) and of these, the role of mannose-BP has been well-studied. As noted above, the amoebae use mannose-BP for cellular attachment, and the fact that only the infective stagetrophozoites but not cysts upregulates mannose-BP expression suggests that, this protein is critical for amoebic invasion (Garate*, et al.*, 2006). However, once the infection is established, microglial cells produce inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β and IL-1α and they can contribute to tissue damage (Benedetto &

The role of the immune system and immune defense mechanisms in protecting against *Acanthamoeba* has not been well characterized, but protection against amoebae appears to involve both innate and adaptive immune responses. Amoebae are extracellular organisms that lack a sialic acid coat or capsule, making them vulnerable to complement-mediated destruction (Bowers & Korn, 1968, Korn & Olivecrona, 1971, Khan, 2005a). Conversely, amoebae can evade immune mechanisms by binding to a C1q component, as shown in the case of *A. culbertsoni*, and the parasite-derived serine proteases can degrade IgG and IgA (Toney & Marciano-Cabral, 1998, Kong*, et al.*, 2000, Na*, et al.*, 2002, Marciano-Cabral & Cabral, 2003). Neutrophils, macrophages, and microglia can destroy amoebae, and their amoebicidal effects are mediated in part by respiratory burst and nitric oxide under the influence of IL-1β, IL-1α, TNF-α and/or IFN-γ (Ferrante, 1991a, Ferrante, 1991b, Marciano-Cabral & Toney, 1998, Marciano-Cabral*, et al.*, 2000, Benedetto & Auriault, 2002a, Benedetto

Affected patients, including healthy individuals upto 90%, carry the *Acanthamoeba*-reactive antibodies of IgM, IgG, and IgA isotypes with no significant differences between males (86.2%) and females (89.2%), indicating that humans are regularly exposed to *Acanthamoeba* and become sensitized with the amoebic antigens (Chappell*, et al.*, 2001, McClellan*, et al.*, 2002, Schuster, 2002, Khan, 2005a, Brindley*, et al.*, 2009, da Rocha-Azevedo*, et al.*, 2009). It has been reported that T cells from healthy individuals can react to *Acanthamoeba* antigens obtained from CSF and antigen-specific T cell clones capable of producing IFN-γ also have

Auriault, 2002, Benedetto*, et al.*, 2003, Marciano-Cabral & Cabral, 2003).

**4. Immune responses to** *Acanthamoeba*

& Auriault, 2002b, Dudley*, et al.*, 2007, Khan, 2008).

(Khan, 2007).

*et al.*, 2009). These two modes of entry lead to the localization of amoebae in the cerebrum and cerebrospinal fluid (CSF) respectively (Elsheikha & Khan, 2010).

Fig. 1. Proposed routes of entry of *Acanthamoeba* into the CNS. Under favorable conditions, cysts become trophozoites which are the infective stages of *Acanthamoeba*. Upon nasal exposure, trophozoites can reach CNS hematogenously via lungs or through olfactory neuroepithelium. The amoeba that gains entry through the skin can also reach CNS hematogenously. However, actual entry into the brain tissue involves crossing the BBB which appears to be mediated through either contact-dependant mechanisms by inducing apoptosis of the endothelial cells or contact-independent mechanisms via destruction of extracellular matrix by amoebic proteases. The micrographs of *Acanthamoeba* trophozoites and cysts were kindly provided by Dr. Francine Marciano-Cabral, Virginia Commonwealth University, VA, USA

Regardless of route of entry, the amoebae have to cross the BBB, either paracellularly by damaging the tight junctions, or transcellularly, in which the integrity of the BBB is maintained (Khan, 2003, Khan, 2006, Khan, 2007). Disruption of the BBB is thought to be mediated by contact-dependent or contact-independent mechanisms (Khan, 2003, Khan, 2006, Khan, 2007). Contact-dependent mechanisms require attachment of the amoeba to the brain's microvascular endothelial cells through amoebic mannose-binding protein (mannose-BP) causing apoptosis of the endothelial cells, a phenomenon that depends on the phosphatidylinositol 3-kinase signaling pathway (Sissons*, et al.*, 2005, Khan, 2006, Khan, 2007). In contrast, contact-independent mechanisms involve extracellular proteases secreted by *Acanthamoeba*, particularly serine proteases, and these enzymes destroy extracellular matrix proteins comprised of collagen (type I, III, and IV), elastin, and fibronectin (Khan*, et al.*, 2000, Sissons*, et al.*, 2005, Khan, 2007). Alternatively, infected immune cells, most importantly macrophages, may simply act as Trojan horses to carry the amoebae to the CNS (Khan, 2007).

The pathogenicity of *Acanthamoebae* varies by species, depending on their inherent potential to tolerate temperatures, attachment to cellular surfaces, and induction of cytolysis (Marciano-Cabral & Cabral, 2003, Khan, 2006). The principal virulent factors are mannose-BP, Nicotinamide adenine dinucleotide (NADH)-dehydrogenase, GDP-mannose pyrophosphorylase and proteasomal ATPase (Marciano-Cabral & Cabral, 2003, Han*, et al.*, 2006) and of these, the role of mannose-BP has been well-studied. As noted above, the amoebae use mannose-BP for cellular attachment, and the fact that only the infective stagetrophozoites but not cysts upregulates mannose-BP expression suggests that, this protein is critical for amoebic invasion (Garate*, et al.*, 2006). However, once the infection is established, microglial cells produce inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β and IL-1α and they can contribute to tissue damage (Benedetto & Auriault, 2002, Benedetto*, et al.*, 2003, Marciano-Cabral & Cabral, 2003).
