**2. Central nervous systems infections due to free living amoebae**

The waterborne disease Primary Amoebic Meningoencephalitis (PAM) was discovered in Australia in the 1960s. Since then, it has been reported from about 15 other countries in Africa, Asia, Europe and North and South America. PAM is caused by *Naegleria fowleri*, and follows intranasal infection during swimming in warm, contaminated freshwater. Most victims have been children or young adults and the disease is almost invariably fatal. Infections have been linked with warm waters such as above-ground pipelines, tropical lakes, geothermal water, heated swimming pools or discharges of industrial cooling water. Until recent cases in the USA were identified, Australia was the only country where *Naegleria fowleri* has been associated with public water supplies (Visvesvara et al., 2007; Heggie, 2010). Recently, the causal agent of a PAM case in the US was diagnosed as *Paravahlkampfia francinae* n. sp., a new species of the free-living amoeba genus *Paravahlkampfia* that was isolated from the cerebrospinal fluid of a patient with headache, sore throat, and vomiting, presenting typical symptoms of PAM caused by *Naegleria fowleri*. Thus awareness of novel emerging amoebae as causative agents of PAM should also be considered (Visvesvara et al., 2009).

PAM should be suspected in young adults and children with acute neurological symptoms as described below and recent exposure to fresh water. The time from initial contact (swimming, diving, water skiing, or simply immersing head in water) to onset of illness is usually 5–7 days, and may even be as short at 24 h. Because there are no distinctive clinical features that differentiate PAM from acute pyogenic or bacterial meningoencephalitis it is imperative that the attending physician obtains information regarding the patient's contact with fresh water, including hot springs, during the past week. The earliest symptoms are sudden appeareance of headaches, high temperature, nuchal rigidity, followed by nausea, vomiting, irritability and restlessness. Nuchal rigidity usually occurs with positive Kernig and Brudzinski signs. Photophobia may occur late in the clinical course, followed by neurological abnormalities, including lethargy, seizures, confusion, coma, diplopia or bizarre behaviour, leading to death within a week. Cranial nerve palsies (third, fourth, and sixth cranial nerves) may indicate brain edema and herniation. Intracranial pressure is usually raised to 600 mm H2O or higher. Cardiac rhythm abnormalities and myocardial necrosis have been found in some cases (Martinez, 1980; Visvesvara et al., 2007).

CSF may vary in colour from greyish to yellowish-white, and may be tinged red with a few red cells (250 mm−3) in the early stages of disease. However, as the disease progresses the red blood cell number increases to as high as 24.600 mm−3. The white blood cell count, predominantly polymorphonuclear leukocytes (PMN), also may vary from 300 cells mm−3 to as high as 26000 mm−3. No bacteria are seen. The CSF pressure is usually elevated (300–600 mm H2O). The protein concentration may range from 100 mg per 100 ml to 1000 mg per 100 ml, and glucose may be 10 mg/100 mL or lower (Martinez, 1980; Visvesvara & Maguire, 2006; Visvesvara et al., 2007). The cause of death is usually increased intracranial pressure with brain herniation, leading to cardiopulmonary arrest and pulmonary edema (Martinez, 1980; Visvesvara & Maguire, 2006; Visvesvara et al., 2007).

Many species of *Acanthamoeba* can cause Granulomatous Amoebic Encephalitis (GAE) also known as *Acanthamoeba* Granulomatous Encephalitis (AGE) (Khan, 2006; da Rocha-Azevedo et al., 2009), is a rare, chronic, progressive infection of the CNS that may involve the lungs (da Rocha-Azevedo et al., 2009). AGE is usually associated with an underlying debilitating disease or immune suppressed individuals including HIV-AIDS patients, diabetics,

The waterborne disease Primary Amoebic Meningoencephalitis (PAM) was discovered in Australia in the 1960s. Since then, it has been reported from about 15 other countries in Africa, Asia, Europe and North and South America. PAM is caused by *Naegleria fowleri*, and follows intranasal infection during swimming in warm, contaminated freshwater. Most victims have been children or young adults and the disease is almost invariably fatal. Infections have been linked with warm waters such as above-ground pipelines, tropical lakes, geothermal water, heated swimming pools or discharges of industrial cooling water. Until recent cases in the USA were identified, Australia was the only country where *Naegleria fowleri* has been associated with public water supplies (Visvesvara et al., 2007; Heggie, 2010). Recently, the causal agent of a PAM case in the US was diagnosed as *Paravahlkampfia francinae* n. sp., a new species of the free-living amoeba genus *Paravahlkampfia* that was isolated from the cerebrospinal fluid of a patient with headache, sore throat, and vomiting, presenting typical symptoms of PAM caused by *Naegleria fowleri*. Thus awareness of novel emerging amoebae as causative agents of PAM should also be

PAM should be suspected in young adults and children with acute neurological symptoms as described below and recent exposure to fresh water. The time from initial contact (swimming, diving, water skiing, or simply immersing head in water) to onset of illness is usually 5–7 days, and may even be as short at 24 h. Because there are no distinctive clinical features that differentiate PAM from acute pyogenic or bacterial meningoencephalitis it is imperative that the attending physician obtains information regarding the patient's contact with fresh water, including hot springs, during the past week. The earliest symptoms are sudden appeareance of headaches, high temperature, nuchal rigidity, followed by nausea, vomiting, irritability and restlessness. Nuchal rigidity usually occurs with positive Kernig and Brudzinski signs. Photophobia may occur late in the clinical course, followed by neurological abnormalities, including lethargy, seizures, confusion, coma, diplopia or bizarre behaviour, leading to death within a week. Cranial nerve palsies (third, fourth, and sixth cranial nerves) may indicate brain edema and herniation. Intracranial pressure is usually raised to 600 mm H2O or higher. Cardiac rhythm abnormalities and myocardial

necrosis have been found in some cases (Martinez, 1980; Visvesvara et al., 2007).

1980; Visvesvara & Maguire, 2006; Visvesvara et al., 2007).

CSF may vary in colour from greyish to yellowish-white, and may be tinged red with a few red cells (250 mm−3) in the early stages of disease. However, as the disease progresses the red blood cell number increases to as high as 24.600 mm−3. The white blood cell count, predominantly polymorphonuclear leukocytes (PMN), also may vary from 300 cells mm−3 to as high as 26000 mm−3. No bacteria are seen. The CSF pressure is usually elevated (300–600 mm H2O). The protein concentration may range from 100 mg per 100 ml to 1000 mg per 100 ml, and glucose may be 10 mg/100 mL or lower (Martinez, 1980; Visvesvara & Maguire, 2006; Visvesvara et al., 2007). The cause of death is usually increased intracranial pressure with brain herniation, leading to cardiopulmonary arrest and pulmonary edema (Martinez,

Many species of *Acanthamoeba* can cause Granulomatous Amoebic Encephalitis (GAE) also known as *Acanthamoeba* Granulomatous Encephalitis (AGE) (Khan, 2006; da Rocha-Azevedo et al., 2009), is a rare, chronic, progressive infection of the CNS that may involve the lungs (da Rocha-Azevedo et al., 2009). AGE is usually associated with an underlying debilitating disease or immune suppressed individuals including HIV-AIDS patients, diabetics,

**2. Central nervous systems infections due to free living amoebae** 

considered (Visvesvara et al., 2009).

individuals undergoing organ transplants or cancer chemotherapy, and drug abusers (Khan, 2006; Visvesvara et al., 2007; da Rocha-Azevedo et al., 2009).

Therefore, the onset of AGE is slow and subtle and develops as a chronic disease from several weeks to months (Visvesvara & Maguire, 2006, Visvesvara et al. 2007). The usual features of AGE consist of headache, stiff neck, and mental-state abnormalities, as well as nausea, vomiting, low-grade fever, lethargy, cerebellar ataxia, visual disturbances, hemiparesis, seizures and coma. Facial palsy with numbness resulting in facial asymmetry is often seen. Cerebral hemispheres are usually the most heavily affected CNS tissue. They are often edematous, with extensive hemorrhagic necrosis involving the temporal, parietal, and occipital lobes. Computerized tomography (CT) scans of the brain show large, low-density abnormalities mimicking a single or multiple space-occupying mass. Magnetic resonance imaging (MRI) with enhancements shows multiple, ring-enhancing lesions in the brain (Seijo-Martínez et al., 2000; Shirwadkar et al. 2006; Visvesvara et al., 2007).

Acanthamoebae infecting the CNS are not readily found in the cerebrospinal fluid (CSF), although they have been isolated from the CSF in a few cases. *Acanthamoeba* that had apparently entered from the nasopharynx through a fistula have been detected in the CSF of a patient without CNS disease (Petry et al., 2006). CSF examination in general reveals lymphocytic pleocytosis with mild elevation of protein and normal or slightly depressed glucose. Examination of the autopsied brain reveals cerebral edema, areas of cortical and basal ganglia softening, and multiple necrotic and hemorrhagic areas of CNS tissues. The brainstem, cerebral hemispheres and cerebellum may show areas of hemorrhagic infarcts. Histological examination reveals the presence of multinucleated giant cells in the cerebral hemispheres, brain stem, mid-brain, cerebellum, and basal ganglion. Necrotic tissue with lipid-containing macrophages and neovascularization suggesting a tumour is often seen. Trophozoites and cysts of acanthamoebae are usually spread all over the infected tissue. Many blood vessels are thrombotic with fibrinoid necrosis and cuffed by polymorphonuclear leukocytes, amoebic trophozoites, and cysts. Multinucleated giant cells forming granulomas may be seen in immunocompetent patients but less often in immunocompromised patients.

Some infected individuals, mostly with HIV/AIDS, develop chronic ulcerative skin lesions, abscesses, or erythematous nodules (Seijo-Martínez et al., 2000; Visvesvara & Maguire, 2006, Visvesvara et al., 2007), especially of the chest and limbs. These nodules are usually solid but sometimes they become ulcerated and purulent. The prodromal period is unknown and several weeks or months may elapse following infection before the disease becomes apparent. Because of the time delay, the precise portal of entry is not clearly known, but the wide dissemination of these amoebae in the environment allows for many possible contacts and modes of infection. Trophic amoebae and/or cysts of *Acanthamoeba* have been isolated from the nasal mucosa of healthy individuals, suggesting a nasopharyngeal route as one means of invasion. Amoebae may also enter the body through ulcers in the skin, resulting in hematogenous dissemination to the lungs and brain, or by inhalation of amoebic cysts (Visvesvara & Maguire, 2006; Visvesvara et al 2007).

In addition to causing CNS infections, *Acanthamoeba* also causes a vision-threatening disease, *Acanthamoeba* **keratitis** (AK) which mostly affects contact lens wearers although many cases have been reported worldwide in non contact lens users mostly related to a previous corneal trauma [6, 7, 8]. The number of affected individuals is increasing worldwide. Morever, recent outbreaks of *Acanthamoeba* keratitis have been recently reported in the United States and Australia (Verani et al., 2009; Tu and Joslin, 2010; Patel et al., 2010;

Encephalitis Due to Free Living Amoebae: An Emerging Issue in Human Health 333

**Treatment of AGE** is problematic because of the lack of clear-cut symptoms, the lack of a good reliable diagnostic test, and the fact that diagnosis is often made postmortem. However, several patients with GAE caused by *Acanthamoeba* spp., as well as some with *Acanthamoeba* cutaneous infection without CNS involvement, have been successfully treated with a combination of pentamidine isethionate, sulfadiazine, flucytosine, and fluconazole or itraconazole. For *Acanthamoeba* cutaneous infection without CNS involvement, topical applications of chlorhexidine gluconate and ketoconazole cream in addition to the abovenoted antimicrobials have resulted in therapeutic success. In many cases, however, therapy had to be discontinued because of undesirable side effects of the medications Visvesvara et al., 2007. A combination of factors –late diagnosis, suboptimal efficacy of antimicrobial therapy, and problems inherent to the immunocompromised host –make for a poor

**Treatment of** *Acanthamoeba* **keratitis** has been fairly successful. A variety of drugs have been used, including chlorhexidine, polyhexamethylene biguanide, propamidine isethionate, dibromopropamidine isethionate, neomycin, paromomycin, polymyxin B, clotrimazole, ketoconazole, miconazole, and itraconazole (Visvesvara et al., 2007; da Rocha-Azevedo, 2009). Brolene, a commercially available eye medication (in the UK, and other EU countries) containing propamidine isethionate and dibromopropamidine isethionate, was found to be effective in the treatment of *Acanthamoeba* infections but may be accompanied by

A number of compounds, including a variety of diamidine compounds, synthetic maganins combined with silver nitrate, imidazole and triazole compounds, azithromycin, phenothiazines and povidone-iodine have been screened *in vitro* for efficacy against *Acanthamoeba* spp. Another drug, miltefosine, an alkylphospholipid, has also been shown to have amoebicidal potential. Significantly, medical cure has been achieved with the application of either polyhexamethylene biguanide (PHMB) or chlorhexidine gluconate with or without Brolene. When medical treatment failed, a combination of debridement and penetrating keratoplasty has been used with good results in some cases. Currently, the drugs of for AK are chlorhexidine gluconate, PHMB and Brolene, and they have greatly improved the prognosis for AK sufferers (reviewed in Visvesvara et al., 2007). However, it was recently demonstrated that clinical strains of *Acanthamoeba* are resistant to the concentrations of chlorhexidine gluconate or PHMB present in contact lens maintenance/disinfection solutions (Martín-Navarro et al., 2008). Thus, the latter together with the demonstrated toxicity of these molecules is supporting the need for novel therapies

*Balamuthia* amoebic encephalitis (BAE) is a rare, subacute to chronic disease that is characterized by hemorrhagic necrotizing lesions or brain abscess (normally detected by neuroimaging scans) with severe meningeal irritation and encephalitis. The lesions are mainly detected in the basal ganglia, midbrain, brainstem and cerebral hemiparesis with characteristic lesions in the CNS parenchyma. Typically, encephalitis is of the granulomatous type composed of the amoebae, CD4 and CD8 T cells, B lymphocytes, few plasma cells, macrophages and multinucleate giant cells (Martinez and Visvesvara, 1997;

**3. Therapy and prognosis of** *FLA* **infections** 

**3.1** *Acanthamoeba* **spp.** 

prognosis for GAE patients.

to treat AK worldwide.

**3.2** *Balamuthia mandrillaris*

drug toxicity and development of resistance.

Joslin et al., 2010). AK is an acute, painful infection that can occur in immunocompetent individuals. When AK is not treated promptly, loss of visual acuity and blindness can occur [6, 7, 8]. Initial symptoms of AK are not specific and include disproportional eye pain, photophobia, eye redness, and tearing, usually affecting one eye. Using a slit-lamp, corneal inflammation leading to formation of a ring-like stromal infiltrate can be observed. Furthermore, corneal epithelial erosion, irregularities, and edema are present. The radial perineural distribution of the infiltrate (radial keratoneuritis) is characteristic for AK, similar to the type of infiltration observed in *Pseudomonas aeruginosa* keratitis. Later stages of infection can result in epithelial denudation and stromal necrosis. Contact lens usage and/or incidents of corneal trauma are strong indicators for AK. Despite the clinical picture, AK is often misdiagnosed as herpes or bacterial keratitides which present similar clinical symptomatology (Khan, 2006; Visvesvara et al., 2007; Martín-Navarro et al., 2008; da Rocha-Azevedo et al., 2009).

Both *Acanthamoeba* and *Balamuthia* cause infections of the lungs and skin (Khan, 2006; Maciver, 2007; Visvesvara et al., 2007; da Rocha-Azevedo et al., 2009). More recently, *Balamuthia mandrillaris*, has been discovered to cause a fatal encephalitis in humans (Maciver, 2007; Visvesvara et al., 2007; Matin et al., 2008). This encephalitis is known as *Balamuthia* amoebic encephalitis (BAE). There are worrying features of BAE that are emerging, even compared to AGE and PAM. PAM is restricted to bodies of warm freshwater, such as swimming pools and lakes, and so can be avoided after its presence has been identified.

AGE is mostly a disease of the immunocompromised, and so affects a small subpopulation of individuals who could conceivably be monitored for early signs of AGE; for example, by inspection of cerebrospinal fluid (CSF) (Deetz et al., 2003; Maciver, 2007). Present data indicate that BAE is more difficult to detect, as it is sporadic, affecting both immunocompromised and immunocompetent, otherwise healthy, individuals with little evidence of predisposing factors (i.e. working in farms or soil related environments. The unpredictable nature of the disease may mean that BAE is even less likely to be diagnosed in time for medical intervention and, like AGE and PAM, it is essential for BAE to be diagnosed early if it is to be treated successfully (Deetz et al., 2003; Petry et al., 2006; Maciver, 2007]. Worryingly, BAE may be relatively a common type of amoebic encephalitis and some cases reported to be due to *Acanthamoeba* have subsequently been shown to be due to *B. mandrillaris* (Deetz et al., 2003; Maciver, 2007).

Recently, *Sappinia diploidea* and *Sappinia pedata* species, also belonging to the free-living amoeba group, that normally live in soil contaminated with faeces of elk, bison, and cattle, have been identified as causing encephalitis in an otherwise healthy individuals (Gelman et al., 2001; Qvarstrom et al., 2009; Walochnik et al., 2010), indicating that there are probably other amoebae that are capable of causing encephalitis in humans.

A study looking at causes of encephalitis found at least 13. 939 cases of acute encephalitis diagnosed between 1990 and 1999 in California [13, 26]. Amongst these cases, 0.1% was attributed to *Naegleria*, 0.63% to other protozoans, and 34.7% were from unspecified causes. The California Encephalitis Project (CEP) (reviewed in Maciver et al., 2007), identified three fatal cases caused by *Balamuthia* from the 334 patients who met the criteria for CEP. No cases of encephalitis caused by either *Acanthamoeba* or *Naegleria* were seen but immunocompromised patients were excluded from the study [30]. Thus, these data would suggest that in California in the 1990s *Balamuthia* and *Naegleria* each accounted for approximately 0.1% of total encephalitis cases in these studies (reviewed in Maciver et al., 2007).
