**6. Diagnosis**

318 Non-Flavivirus Encephalitis

ACA 83-95, can induce clinical signs of autoimmune encephalomyelitis in the adjuvant protocol of inducing CNS autoimmunity in SJL mice (Massilamany*, et al.*, 2010, Massilamany*, et al.*, 2011). **(b) Epitope spreading.** *A. castellanii* causes granulomatous inflammation in the brain and spinal cord, which can lead to the local release of myelin antigens and prime T cells. Alternatively, the newly released myelin antigens are carried by antigen-presenting cells to the draining lymph nodes and prime T cells, which, in turn, migrate back into the CNS and further aggravate inflammation. Similar events have been earlier demonstrated in the case of

MS is a disease of the CNS characterized by inflammation and infiltration of MNC and the loss of myelin sheath encapsulating the axons (Noseworthy*, et al.*, 2000, Sospedra & Martin, 2005). Autoimmune responses to myelin antigens have been implicated in MS pathogenesis and this requires the mediation of autoreactive T cells and B cells, but the mechanisms by which the disease is initiated are unknown (Kerlero de Rosbo*, et al.*, 1993, Sospedra & Martin, 2005). Although genetic susceptibility is a major predisposing factor, exposure to environmental microbes such as viruses and bacteria have been suspected in the initiation of autoimmune diseases. In support of the latter, exacerbations of MS attacks or temporal alterations in the disease course have been linked primarily to exposure to virus infections such as Epstein Barr virus and Human Herpes virus-6, but the clinical evidence remains elusive (Cirone*, et al.*, 2002, Pohl, 2009, Salvetti*, et al.*, 2009). The current dogma is that MS does not appear to follow Koch's postulates in that no single organism appears to trigger it; rather, exposure to multiple organisms might be critical for MS predisposition (Sospedra & Martin, 2005). The fact that ACA contains mimicry epitope for PLP, one of the candidate autoantigens implicated in MS pathogenesis, suggests that ACA infection can potentially

*Acanthamoeba*-induced encephalitis is often overlooked (Schuster & Visvesvara, 2004, da Rocha-Azevedo*, et al.*, 2009), partly due to the rarity of *Acanthamoeba* infections and a lack of familiarity and diagnostic tools. However, when diagnosed, it is difficult to differentiate PAM from GAE because symptoms overlap between each other (da Rocha-Azevedo*, et al.*, 2009). PAM is initially manifested by severe headache, rhinitis, nausea, and fever followed by anosmia, seizures, stiff neck, diplopia, and coma, finally leading to death (Marciano-Cabral & Cabral, 2003, da Rocha-Azevedo*, et al.*, 2009). Histologically, brains contain inflammatory infiltrates comprised of neutrophils, eosinophils, and macrophages (Martinez & Janitschke, 1985, Marciano-Cabral & Cabral, 2003, da Rocha-Azevedo*, et al.*, 2009). In contrast, symptoms of GAE are diverse in that a wide range of clinical manifestations can be expected. These include headache, rise in intracranial pressure, abnormal gait or ataxia, diplopia, stiff neck, confusion, behavioral changes, hemiparesis, cranial nerve palsies, seizures, photophobia, and anorexia; more than 90% of individuals affected with GAE tend to die (Marciano-Cabral & Cabral, 2003, Khan, 2006). The histologic disease is characterized by hemorrhagic or necrotic encephalitis, edema of the brain accompanied by the presence of focal lesions around the cerebrum, cerebellum, and corpus callosum. Cellular infiltrations in histological sections include multinucleated giant cells, plasma cells, polymorphonuclear cells and mononuclear cells (Martinez & Visvesvara, 1997, da Rocha-Azevedo*, et al.*, 2009) and HIV patients can develop granulomas within the CNS possibly due to low CD4 T cell count (Marciano-Cabral & Cabral, 2003, Cha*, et al.*, 2006, Khan, 2006). In some patients,

murine pathogen, TMEV (Miller*, et al.*, 2001, Olson*, et al.*, 2004, McMahon*, et al.*, 2005).

lead to the generation of PLP reactive T cells and predispose to MS.

**5. Clinical signs and histology** 

*Acanthamoeba*-induced encephalitis is not routinely suspected, the disease can be misdiagnosed as neurocysticercosis; viral, rickettsial, fungal, and bacterial meningitis; toxoplasmosis; and brain tumors (Schuster & Visvesvara, 2004, khan, 2005b). Serologically, detection of *Acanthamoeba*-reactive antibodies gives an indication of amoebic exposure at a population level (Cursons*, et al.*, 1980, Cerva, 1989, Khan, 2006). However, definitive diagnosis requires the demonstration of amoebic trophozoites or cysts in biological samples. Examination of wet-mount smears prepared from CSF or methanol-fixed smears stained with Giemsa-Wright permit identification of amoebic trophozoites. While evaluating wetmount smears, careful consideration should be given to differentiate trophozoites from macrophages because of their close morphological resemblance to each other (Cleland*, et al.*, 1982, Lalitha*, et al.*, 1985, Singhal*, et al.*, 2001). In addition to CSF, trophozoites can also be detected in bronchoalveolar lavage fluid from patients with respiratory distress (Newsome*, et al.*, 1992). Detection of trophozoites in fixed tissue sections prepared from brains is usually performed using hematoxylin and eosin and trichrome stainings (Newsome*, et al.*, 1992). In contrast, amoebic cysts in brain tissues are detected using calcofluor white staining (Silvany*, et al.*, 1987). Alternatively, periodic acid-Schiff's stain and Gomori-methenamine silver stain can be used to stain tissue sections in which, cysts appear red whereas tissues appear black in color (Marciano-Cabral & Cabral, 2003).

Other specialized techniques employed to demonstrate the presence of amoeba are transmission electron microscopy and immunofluorescent or immunoperoxidase staining (Willaert & Stevens, 1976, Stevens*, et al.*, 1977, McKellar*, et al.*, 2006, Guarner*, et al.*, 2007). However, because most *Acanthamoeba* spp. are antigenically related, the use of immunohistochemical techniques does not permit identification by species. To identify structural brain lesions, computed tomography and magnetic resonance imaging are widely used (Sell*, et al.*, 1997, Kidney & Kim, 1998). These evaluations can reveal changes such as multifocal areas of signal intensities or ring-like lesions or low-density areas indicating occupying mass of tumor or abscess (Martinez*, et al.*, 1977, Martinez*, et al.*, 1980, Ofori-Kwakye*, et al.*, 1986, Matson*, et al.*, 1988, khan, 2005b, Khan, 2008, da Rocha-Azevedo*, et al.*, 2009). The regions of the brain that are usually affected are midbrain, basal areas of the temporal and occipital lobes, and the posterior fossa (Seijo Martinez*, et al.*, 2000, Marciano-Cabral & Cabral, 2003, Khan, 2006). Molecularly, PCR amplification of the 18S rDNA using sequence-specific primers is currently used as a quick and reliable method of diagnosis (Schroeder*, et al.*, 2001, Khan, 2006, da Rocha-Azevedo*, et al.*, 2009, Maritschnegg*, et al.*, 2011). Hematologically, pleocytosis accompanied by lymphocytosis, neutrophilia, hypoglycemia and hyperprotenemia may be seen in patients with GAE (Marciano-Cabral & Cabral, 2003). In addition, *Acanthamoeba* can be isolated from clinical specimens by plating the samples on non-nutrient agar plates coated with *E. coli* or *Enterobacter aerogenes*  (Schuster, 2002, Khan, 2006, da Rocha-Azevedo*, et al.*, 2009).
