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

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

332 Non-Flavivirus Encephalitis

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-

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

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

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

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

Azevedo et al., 2009).

been identified.

to *B. mandrillaris* (Deetz et al., 2003; Maciver, 2007).

other amoebae that are capable of causing encephalitis in humans.

encephalitis cases in these studies (reviewed in Maciver et al., 2007).

**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 prognosis for GAE patients.

**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 drug toxicity and development of resistance.

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 to treat AK worldwide.

#### **3.2** *Balamuthia mandrillaris*

*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;

Encephalitis Due to Free Living Amoebae: An Emerging Issue in Human Health 335

*N. fowleri* (Visvesvara et al., 2007; da Rocha-Azevedo et al., 2009). Azithromycin, a macrolide antimicrobial, has been shown to be effective against *Naegleria* both in vitro and in vivo (mouse model of disease). However, other macrolides (erythromycin, clarithromycin) are less effective. *Naegleria fowleri* is also sensitive to voriconazole (Marciano-Cabral and Cabral,

As it has been recently reported as pathogenic to humans and other animals, no studies

*Balamuthia mandrillaris (BAE)* 

and cyst

Two stages: amoeba

Vesicular nucleus with single or multiple nucleoli; amoeboid and 'spider-like' movements in culture; cyst wall with three layers

Infection from soil, water, and air

Immunocompetent (children and elderly)

immunocompromise d individuals; Hispanic Americans

Headache, nausea, seizures, stiff neck, hydrocephalus; sinus infection; nodule formation in cutaneous infections

Indolent subacute course; once in acute stage, fatal in weeks

or

*Sappinia spp.* 

Two stages: amoeba and cyst

Presence of two abutting nuclei in amoeba and cyst stages

Present in soil, water and in air; originally identified from herbivore faeces.

Insufficient data

Insufficient data

Headache, vomiting, photophobia, loss of consciousness; preceded by sinus infection

have been carried out so far regarding therapy and prognosis of this amoeba.

and cyst

pores

*Acanthamoeba (AGE)* 

Two stages: amoeba

Vesicular nucleus; finger-like pseudopodia projecting from surface; cyst wall with two layers and with

*period* Days Weeks to months Weeks to months Insufficient data

Infection from soil, water, and air; present

environment (water taps, hydrotherapy pools, air conditioning cooling towers)

immunocompromised

Headache, stiff neck, behavioural changes,

Indolent subacute course; acute stage fatal in weeks

in hospital

Typically,

individuals

coma

2003; Visvesvara et al., 2007; da Rocha-Azevedo et al., 2009).

*Naegleria fowleri* 

Vesicular nucleus;

Humans typically infected while recreating in warm fresh waters

Children and young adults in good health

Headache, stiff neck, seizures,

coma

Fulminant disease; death within 1–2 weeks without treatment

Three stages: amoeba, cyst and flagellate

*(PAM)* 

limacine movement of amoebae; flagellate stage; cyst with pores flush at the surface

**3.4** *Sappinia* **spp***.*

*Life cycle* 

*Distinctive morphological features* 

*Prodromal* 

*Epidemiology* 

*Groups at risk* 

*Disease at presentation* 

*Clinical course* 

2001; Matin et al., 2008). However, in immunocompromised patients with an impaired cellular immune response, granuloma formation may be minimal or absent (Martinez and Visvesvara, 1997; 2001).

Post mortem examination often shows severe edema and hemorrhagic necrosis. The amoebae colonize the brain tissue and produce subacute necrotising hemorrhagic encephalitis leading to brain dysfunction. *Balamuthia mandrillaris* trophozoites and cysts are present within the perivascular spaces and within the necrotic CNS parenchyma (Martinez and Visvesvara, 1997). The disease is likely to take a cutaneous route before secondarily attacking the CNS. The time period of transition from the cutaneous form to the CNS ranges from 30 days to 2 years, with an average of 5 –8 months (Bravo and Sanchez, 2003). The skin lesions may appear at the site of an abrasion of the skin surface of the patient, or lesions can appear as single or multiple plaques or nodules (Deetz et al., 2003). These plaques may appear on the face, the trunk or the limbs, with a rubbery to hard consistency (Bravo and Sanchez, 2003). Skin lesions indicate a site of entry and are frequently observed in BAE patients.

Because most of the cases of BAE have presented with no clear-cut clinical profile, they have been treated empirically with steroids as well as with antibacterial, antifungal and antiviral agents with almost no effect upon the course of the infection. Anti-inflammatory steroids that were administered may have actually facilitated spread of the infection by suppressing the inflammatory response. Two patients from California, a 60-year-old man, and a 6-year old girl, in addition to a 70-year-old woman from New York survived balamuthiasis after treatment with a combination of pentamidine isethionate, sulfadiazine, clarithromycin, fluconazole, and flucytosine (5-fluorocytosine) (Deetz et al., 2003).

In the case of the Peruvian balamuthiasis patients with cutaneous lesions (Martínez et al., 2010; Bravo et al., 2011), one recovered without any treatment and two others became well after prolonged therapy with albendazole and itraconazole. The use of multiple antimicrobials in treatment makes it difficult to single out one or more of the drugs that might be the basis for optimal therapy. Furthermore, drugs may show synergistic activities in vivo that are not seen in in vitro testing. In vitro studies have shown that pentamidine and propamidine isethionates were amoebastatic but not amoebicidal. Among other drugs tested with little or no activity were macrolide antibiotics, azole compounds, gramicidin, polymyxin B, trimethoprim, sulfamethoxazole, and a combination of trimethoprimsulfamethoxazole as well as amphotericin B (Maciver, 2007; Visvesvara et al., 2007).

Recent information based on in vitro data has shown that miltefosine was able to lysed the amoebae. Voriconazole, however, had virtually no effect on *Balamuthia* (Maciver, 2007; Visvesvara et al., 2007). Given the problems with the diagnosis of infection and the lack of effective antimicrobial agents, the prognosis for patients is poor.

#### **3.3** *Naegleria fowleri*

Few patients have survived PAM. One of these survivors, a Californian girl, was aggressively treated with intravenous and intrathecal amphotericin B, intravenous and intrathecal miconazole, and oral rifampin [2, 28]. Over a 4- year follow-up, she remained completely healthy and free of any neurological deficits. It was believed that amphotericin B and miconazole had a synergistic effect but that rifampin was without effect on the amoebae. 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 CNS, were found to have inhibitory effects on *N. fowleri* (Visvesvara et al., 2007; da Rocha-Azevedo et al., 2009). Azithromycin, a macrolide antimicrobial, has been shown to be effective against *Naegleria* both in vitro and in vivo (mouse model of disease). However, other macrolides (erythromycin, clarithromycin) are less effective. *Naegleria fowleri* is also sensitive to voriconazole (Marciano-Cabral and Cabral, 2003; Visvesvara et al., 2007; da Rocha-Azevedo et al., 2009).
