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

Free-living amoebae (FLA) belonging to Acanthamoeba and Sappinia genera as well as Balamuthia mandrillaris and Naegleria fowleri species are aerobic, mitochondriate, eukaryotic protists that occur worldwide and can potentially cause infections in humans and other animals (Visvesvara and Maguire, 2006; Visvesvara et al., 2007). Due to the fact that these amoebae have the ability to exist as free-living organisms in nature and only occasionally invade a host and live as parasites within host tissue, they have also been called amphizoic amoebae (Page, 1988). All four amoebae are known so far to cause infections of the central nervous system (CNS). Several species of Acanthamoeba (i.e. A. castellanii, A. culbertsoni, A. hatchetti, A. polyphaga, A. rhysodes), the only known species of Balamuthia, B. mandrillaris, two species of Sappinia genus, S. diploidea and S.pedata, and only one species of Naegleria, N. fowleri, are known to cause disease in humans and other animals (Khan, 2006; Visvesvara et al., 2007).

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;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).

Therapy and prognosis of FLA infections
3.1 Acanthamoeba spp.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.

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;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.

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).

Sappinia spp.
As it has been recently reported as pathogenic to humans and other animals, no studies have been carried out so far regarding therapy and prognosis of this amoeba.

Novel therapeutic approaches
Recently, the application of siRNA in Acanthamoeba species (Lorenzo-Morales et al., 2005;2008;2010) has opened a novel approach for the progress of future therapies based on siRNAs alone or in combination with chemical compounds.Also the use of RNAi moleculaes could be very powerful for the identification of novel drug targets and metabolic pathways in these pathogens that could be exploited for the development of new therapeutic agents.Recently, RNAi methodology has been successfully use in Naegleria fowleri (Jung et al., 2008) resulting in a reduced pathogenicity of the RNAi-treated amoebae.Therefore, RNAi molecules are currently presenting as very powerful tools that are waiting to be fully exploited in the development of new therapies against pathogenic FLA.

Conclusions
Regarding encephalitis caused by FLA, it is important to mention that because most of them are ultimately fatal, diagnosis of these infec t i o n s i s o f t e n m a d e a t a u t o p s y , e v e n i n developed countries where sophisticated diagnostic facilities are readily available.However, in Sub-Saharan Africa and Southeast Asia, where HIV/AIDS rates are increasing, it is quite possible that a large number of cases have gone undetected.A similar case occurs in South America, where skin infections due to B. mandrillaris are a common episode in countries like Peru but the lack of diagnostic and treatment facilities in most areas have caused that most of these cases have been unnoticed as well.This due to a number of reasons: (1) lack of expertise to identify these pathogenic amoebae; (2) cultural methods and expense that prevent autopsies; and (3) an abundance of more prominent diseases such as HIV/AIDS, tuberculosis and malaria that consume national and international resources.Therefore, the actual incidence of the encephalitis caused by FLA is not really known although it seems that these pathogenic amoebae are emerging as causative agents of encephalitis worldwide.