Balamuthia mandrillaris: Morphology, biology, and virulence.

Balamuthia mandrillaris is a protist pathogen that can cause encephalitis with a fatality rate of >95%. This is due to our incomplete understanding of the pathogenesis and pathophysiology of B. mandrillaris encephalitis. B. mandrillaris has two stages in its life cycle, an active trophozoite stage during which it divides mitotically. However, under unfavorable conditions, the trophozoite transforms into a dormant cyst stage. A major concern during the course of therapy is that B. mandrillaris can transform into cysts. Cysts are highly resistant to physical and chemical conditions and present a problem in successful antimicrobial chemotherapy. Several lines of evidence suggest that B. mandrillaris encephalitis develops as a result of hematogenous spread, but it is unclear how circulating amoebae enter the central nervous system and cause inflammation, blood-brain barrier disruption, and neuronal injury. Recent studies have identified several parasite-host determinants for B. mandrillaris translocation of the blood-brain barrier, and host inflammatory markers that may be associated with neuronal injury. These determinants may provide important targets for the prevention and treatment of this devastating infection. Here, we present a brief overview of the current understanding of the morphology, biology, pathogenesis, and pathophysiology of B. mandrillaris encephalitis.

INTRODUCTION

Balamuthia mandrillaris is an emerging protist pathogen; a free-living amoeba that was initially encountered in 1986 from the brain necropsy of a mandrill baboon (Papio sphinx) who died of a neurological disease at the San Diego Zoo Wild Animal Park, California, USA[1] (Visvesvara et al., 1990). This protist pathogen produced necrotizing hemorrhagic encephalitis similar to the granulomatous encephalitis caused by another free-living amoeba, Acanthamoeba. Although closely related to Acanthamoeba, based on light and electron microscopic studies, animal pathogenicity testing, antigenic analyses and ribosomal RNA sequences, a new genus, Balamuthia[234] was created (Visvesvara and Stehr-Green, 1990; Amaral Zettler et al., 2000; Booton et al., 2003). B. mandrillaris is now known to cause serious cutaneous infections and fatal encephalitis involving the central nervous system (CNS). So far, more than 200 cases have been reported worldwide[567] (Lorenzo-Morales et al., 2013, Bravo and Seas, 2012 and Cary et al., 2010), however, the true burden of this infection remains unknown. The majority of B. mandrillaris encephalitis cases are reported from the Americas; this may be due to greater research interests on the part of investigators in this region. As physicians and researchers elsewhere become more familiar with this pathogen, patterns may change in the future[5] (Lorenzo-Morales et al., 2013). Distressingly, fatality rates have been estimated to approach 98%, and no specific successful treatment has yet been reported for this infection. At present, the predisposing factors for B. mandrillaris encephalitis remain incompletely understood. What's more, unlike Acanthamoeba; Balamuthia can produce encephalitis in relatively immunocompetent individuals, leading almost always to death, and consequently presents a significant threat to human health. This is of particular concern in view of (i) The increasing numbers of immunocompromised patients (ii) the excessive use of antibiotics (iii) global warming with increased outdoor activities adding to the ubiquity of these pathogens, thus increased exposure to the susceptible hosts etc. Perhaps, the most distressing aspect is the inadequate availability of effective and/or recommended treatment against B. mandrillaris encephalitis. The purpose of this review was to provide our current understanding of B. mandrillaris morphology and biology, the disease, virulence, and pathogenesis of this infection.

BIOLOGY AND CELLULAR MORPHOLOGY OF BALAMUTHIA MANDRILLARIS

Because of its recent discovery, our current knowledge of this protist pathogen is very limited. B. mandrillaris has two stages in its life cycle, a vegetative trophozoite stage and a dormant cyst stage [Figure 1]. The trophozoite stage is approximately 30–60 μm in diameter that contains a single nucleus (although binucleated forms have been observed). The nucleus may possess more than one nucleolus, which may aid in its differentiation from its close relative: Acanthamoeba. The trophozoites contain numerous mitochondria, ribosomes, endoplasmic reticulum, and divide by asexual reproduction that occurs by binary fission. The trophozoites form broad pseudopodia and exhibit filamentous[8910] structures (Schuster and Visvesvara, 2004; Jayasekera et al., 2004; 2005). Under harsh conditions (lack of food, extremes in osmolarity, pH and temperatures, increasing density of parasite populations), the amoebae switch into a dormant cyst stage a process known as “encystment.” Encystment ensures the survival of the organism in adverse environmental conditions. The cysts of B. mandrillaris are spherical, uninucleate, and are approximately 10–30 μm. Transmission electron microscopic studies show that cysts in B. mandrillaris have three walls: An inner wall, that is, endocyst and an outer wrinkled wall, that is, ectocyst. Both layers are separated with an undefined thick amorphous, fibrillar middle layer, the mesocyst[11] (Martinez et al., 2001) [Figure 2]. Only two layers, however, are detected when the cysts are observed under the optical microscope. The trophozoites emerge from the cysts under favorable conditions and actively reproduce, thus completing the life cycle [Figure 1].

Figure 1

The life cycle of Balamuthia mandrillaris. Under favorable conditions, B. mandrillaris remains in the active trophozoites form, as observed under phase-contrast microscope (×250) exhibiting distinct filamentous characteristics. Under harsh conditions, trophozoites differentiate into round cysts (×250)

Figure 2

Transmission electron micrograph of a Balamuthia mandrillaris cyst. The wall is made up of a thin, wavy ectocyst, a fibrous mesocyst, and a thick round endocyst. The cytoplasm is filled with numerous pinocytotic vacuoles and/or vesicles as well as mitochondria

Furthermore, the cysts of B. mandrillaris are highly resistant to physical and chemical conditions. Studies were performed to test the resistance of B. mandrillaris to physical, chemical, and radiological conditions. Following treatments, viability was determined by culturing amoebae on the human brain microvascular endothelial cells (HBMEC) as a food source for up to 12 days. It was found that B. mandrillaris cysts are resistant to repeated freeze–thawing (5 times), temperatures of up to 70°C, 0.5% SDS, 25 ppm chlorine, 10 mg pentamidine isethionate per mL and 200 mJ ultraviolet irradiation cm2[12] (Siddiqui et al., 2008). These studies reveal that B. mandrillaris cysts are resistant[12] to denaturing conditions, and that cyst walls are composed following linkage analyses, at least in part, of carbohydrates (Siddiqui et al., 2008). Examination of the carbohydrate composition of the cyst wall revealed that the major components are mannose (20.9 mol%) and glucose (79.1 mol%) with trace amounts of the galactose present.[13] Linkage analysis shows cyst wall carbohydrates with apparently linear and branching saccharides and suggests the presence of cellulose (Siddiqui et al., 2010).

Recently, the topographic microscopic analysis of B. mandrillaris trophozoites and cysts using[14] atomic force microscope were observed (Aqeel et al., 2014). Atomic force microscopy involves the study of the precise description of shape, surface, and feature of the specimen. B. mandrillaris trophozoites were identified as irregular in structure with no defined shape. The two-dimensional and three-dimensional images reveal that the organism has an uneven surface with a diameter of approximately 10 μm and the mean height of B. mandrillaris is 2.76 μm. On the other hand, B. mandrillaris cysts are circular in shape, with no evidence of projections and the mean diameter of B. mandrillaris cysts is 20 μm, while the mean height of B. mandrillaris cysts is 1.46 μm[14] (Aqeel et al., 2014).

In addition, phase micrographic analysis of B. mandrillaris trophozoites and cysts[14] using atomic force microscope was performed; which measures the stiffness of the sample (Aqeel et al., 2014). For B. mandrillaris trophozoites, no stiffness was recorded (diffused structure) in either two-dimensional or three-dimensional images and no boundary stiffness could be measured. In contrast, for B. mandrillaris cysts, a ring-like structure of hardiness/stiffness in both two-dimensional and three-dimensional images was observed. The diameter of the hard/stiff region is 3.517 μm, which was regularly observed for the whole cyst, most likely representing the endocyst wall of B. mandrillaris cysts.

On a separate note, given the amoebistatic response of B. mandrillaris to the classical antifungal compound, ketoconazole, it is likely that their membranes contain ergosterol[1516] (Sande et al., 1985; Siddiqui et al., 2007). This is not surprising, given that Balamuthia is a close relative of Acanthamoeba, and ergosterol is known to be a major sterol membrane component of Acanthamoeba (Raederstorff et al., 1985; Smith et al., 1968).[1718] Of note, ergosterol biosynthesis is limited to fungi and protozoa while human cells contain cholesterol. Thus, ergosterol biosynthesis can be exploited to interfere with the biological processes of B. mandrillaris without harming the host. B. mandrillaris does not feed on Gram-negative bacteria (as do Acanthamoeba), and thus the use of nonnutrient agar plates coated with bacterial cultures[19] is an ineffective method for its isolation. However, B. mandrillaris grows well on mammalian cell cultures or other eukaryotic cells with a doubling time of 18-50 h, and recently, complex axenic medium has been used for their cultivation (Schuster et al., 1996). There is very little knowledge regarding the ecological distribution of B. mandrillaris.[19] This is due to the fact that these amoebae are difficult to isolate. Up until previously, B. mandrillaris had only been isolated from soil and dust samples (Schuster et al., 2003a; b).[2021] However, recent reports document the isolation of B. mandrillaris from water (Baquero et al., 2014; Lares-Jiménez et al., 2014).[222326]

BALAMUTHIA AMOEBIC ENCEPHALITIS

Balamuthia mandrillaris can cause CNS infection, namely Balamuthia amoebic encephalitis (BAE), and nasopharyngeal or cutanous infections. BAE is rare, subacute to chronic disease that almost always proves fatal. However, as the disease is rare, this suggests the presence of predisposing factors. In addition to immunocompetent individuals, BAE has been reported in patients suffering from cancer or diabetes, human immunodeficiency virus (HIV)-infected patients or drug and alcohol abusers.

CUTANEOUS DISEASE

The cutaneous form of the infection is often overshadowed by the CNS involvement. In this form, 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 Seas, 2006).[24] 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; Bravo and Seas, 2006).[2425] These plaques may appear on the face, the trunk or the limbs, with a rubbery to hard consistency (Bravo et al., 2006).[24]

PATHOPHYSIOLOGY OF BALAMUTHIA AMOEBIC ENCEPHALITIS

Although, BAE can occur in healthy individuals, immunocompromised or debilitated patients due to HIV infection, diabetes, immunosuppressive therapy, malignancies, malnutrition, and alcoholism are particularly at risk[82728] (Schuster and Visvesvara, 2004; Visvesvara et al., 2007; Siddiqui and Khan, 2008). The risk factors for patients suffering from the above diseases include exposure to contaminated water such as swimming pools, on beaches, or working with garden soil. The clinical symptoms resemble viral or bacterial meningitis, leptomeningitis, and tuberculous meningitis and are characterized by headache, fever, characteristic skin lesions, stiff neck, nausea, sleepiness, mood swings, hemiparesis, aphasia, vomiting, acute confused state, cranial nerve palsies, increased intracranial pressure, seizures, brain edema and finally lead to death (Martinez and Visvesvara, 1997; Jayasekera et al., 2004; Schuster and Visvesvara, 2004; White et al., 2004).[892930] The amoebae attack the brain tissue and produce subacute necrotizing hemorrhagic encephalitis leading to brain dysfunction.[29] B. mandrillaris trophozoites and cysts are present within the perivascular spaces and within the necrotic CNS parenchyma (Martinez and Visvesvara, 1997). Typically, encephalitis is of the granulomatous type composed of CD4 and CD8 T-cells, B lymphocytes, few plasma cells, macrophages and multinucleate giant cells (Martinez et al., 2001).[11] However, in immunocompromised patients with an impaired cellular immune response, granuloma formation may be minimal or absent (Martinez et al., 2001).[11]

PATHOGENESIS OF BALAMUTHIA AMOEBIC ENCEPHALITIS

Even though there have been improvements in the diagnosis of BAE (Huang et al., 1999; Booton et al., 2003a, b; Yagi et al., 2005; Qvarnstrom et al., 2006; Tavares et al., 2006),[313233343536] however, the pathogenesis and pathophysiology of this disease remains incompletely understood. The routes of entry include the respiratory tract, leading to amoebae invasion of the intravascular space followed by hematogenous spread. Alternatively skin lesions may provide direct amoebal entry into the bloodstream, [Figure 3]. Of note, are the recent studies using animal models, which show the gastrointestinal tract as a possible route of entry[37] (Kiderlen et al., 2006). Amoebal invasion of the CNS most likely occurs at the sites of the blood-brain barrier[8122938] (Martinez and Visvesvara, 1997; Schuster and Visvesvara, 2004; Matin et al., 2008; Siddiqui et al., 2008). Other routes of entry may include amoebal invasion of the nasal mucosa and migration along nerve fibers, followed by invasion of the olfactory bulb (Kiderlen and Laube, 2004).[39] It is thought that the cutaneous and respiratory infections can lasts for months, but the involvement of the CNS can lead to fatal consequences within days or weeks.

Figure 3

The model of Balamuthia amoebic encephalitis. Amoebae enter the body via the nose and either enter into lungs or travel along the olfactory neuroepithelial route, finally leading to amoebae invasion of the central nervous system

The precise molecular mechanisms of B. mandrillaris transmigration of the blood-brain barrier are not known. Several events may combine to disrupt the blood-brain barrier, including B. mandrillaris adhesion, proteolytic attack, and/or host inflammatory responses leading to blood-brain barrier injury [Figure 4]. Moreover, recent studies in vitro show that B. mandrillaris exhibit binding to HBMEC, which model the blood-brain barrier in a galactose-inhibitable manner thus indicating the presence of a galactose-binding protein on the surface membranes of B. mandrillaris (Matin et al., 2007).[40] Subsequent to initial binding, HBMEC incubated with B. mandrillaris release significantly higher levels (>400 pg/mL) of interleukin-6 (IL-6, a pleiotropic cytokine known to play a key role in initiating early inflammatory responses). The primary function of the inflammatory response is to recruit leukocytes to the site of infection by modulating the expression of adhesion molecules like intercellular adhesion molecule-1. The pro-inflammatory cytokines, tumor necrosis factor-alpha, IL-1 together with IL-6, IL-8, and granulocyte/macrophage colony-stimulating factor have been implicated in the increase of blood-brain barrier permeability (Leib and Tauber, 1999).[41] Furthermore, it is shown that B. mandrillaris-mediated IL-6 release is dependent on phosphatidylinositol 3-kinase activation (Jayasekera et al., 2005).[10] Overall, these studies suggest that B. mandrillaris stimulate the activation of host intracellular signaling pathways leading to inflammatory responses. The cumulative effects of these events eventually lead to blood-brain barrier perturbations as demonstrated by HBMEC cytotoxicity (Jayasekera et al., 2004; Matin et al., 2006).

Figure 4

The direct and indirect virulence factors that most likely contribute to the pathogenesis of Balamuthia amoebic encephalitis

Other recent studies have shown that B. mandrillaris exhibit proteolytic activities to degrade the extracellular matrix (ECM). In healthy brains, the ECM comprises a major percent of the normal brain volume (Gladson, 1999) that forms the basal lamina around the blood vessels as well as providing the critical structural and functional support to the neuronal tissue.[42] The excessive ECM degradation affects neurovascular structural/functional properties that are highly destructive to CNS functions. Thus, the ability of the amoebae to degrade the ECM may aid in their invasion of and growth in the brain tissue (Matin et al., 2006).[43] B. mandrillaris proteases are inhibited with 1, 10-phenanthroline, which indicates that they are metalloproteases in nature. In addition, in vitro studies reveal that live B. mandrillaris hydrolyze extracellular ATP (Matin et al., 2008). This ability of B. mandrillaris to hydrolyze ATP may have a role in the biology and pathogenesis of B. mandrillaris.[38] Future studies are needed to further determine the host susceptibility factors, B. mandrillaris colonization of the skin lesions and/or nasopharynx leading to amoebae entry into the intravascular space, parasite survival within the bloodstream, and invasion of the CNS and the brain tissue damage ultimately leading to meningoencephalitis. This will help to design preventative strategies and/or will identify potential targets for the rationale development of therapeutic interventions.

INDIRECT VIRULENCE FACTORS

As we know, the ability of B. mandrillaris to produce human diseases is a multifactorial process and among other factors, is dependent on their ability to survive outside its mammalian host for various times and under diverse environmental conditions (high osmolarity, varying temperatures, food deprivation, and resistance to chemotherapeutic drugs). It is most likely the cyst stage that allows B. mandrillaris to overcome such conditions. Consequently, the ability of B. mandrillaris to switch its phenotype can be considered as contributory factors toward disease and are indicated as indirect virulence factors.

IMMUNE RESPONSE TO BALAMUTHIA MANDRILLARIS

B. mandrillaris antibodies of the IgG and IgM classes are detected in healthy populations with titers ranging from 1:64 to 1:256. Cord blood also contains antibodies but at lower titers, perhaps the result of cross-placental transfer from the maternal circulatory system.[31] However, the antibody levels are very low in neonates, which suggest that these substantially increase with age, most likely as a result of environmental exposure to the amoeba in the soil (Huang et al., 1999).

Recently, a study revealed that serum exhibited protective effects on B. mandrillaris binding to and the subsequent cytotoxicity of HBMEC (Matin et al., 2007).[40] Normal human serum exhibits an initial limited amoebicidal effect; approximately 40% trophozoites are killed.[44] However, a sub-population of amoeba remains viable but cultures are stationary over longer incubations. The fact that serum exhibits approximately 50% inhibition of amoeba binding to HBMEC (similar to amoebicidal effects) suggests that effects of serum on the properties of B. mandrillaris are at least partly secondary to the amoebicidal/amoebistatic effects. This is consistent with previous findings, which show that virulent strains of Acanthamoeba resists serum-mediated killing (Toney et al., 1998). Another interesting finding is that serum possesses antibodies that react with several B. mandrillaris antigens in Western blotting assays (Matin et al., 2007). The antigens of B. mandrillaris react strongly with normal human serum. B. mandrillaris isolated from the baboon tissue (ATCC 50209) and from the human brain share several common antigens confirming that both isolates are antigenically close and belong to the same species.[40] Overall, these studies suggest that normal human serum is partially adept of inhibiting B. mandrillaris properties associated with its pathogenesis but whether a healthy immune response is sufficient to control and/or eradicate this life-threatening pathogen is uncertain.[9] To this end, studies are being conducted to determine the detrimental effects of serum on B. mandrillaris in the presence of neutrophils/macrophages. These studies will clarify the mechanisms associated with B. mandrillaris pathogenesis, which can help design preventative measures and/or develop therapeutic interventions. Nonetheless, the protective role of antibodies against BAE is somewhat vague. For example, several BAE patients are reported to possess high titer of anti-B. mandrillaris antibodies without a protective response that resulted in death (Jayasekera et al., 2004). This could be due to delayed humoral response, overwhelming BAE infection, or the ability of amoeba to evade the humoral immune response.

CONCLUDING REMARKS

While the number of infections due to B. mandrillaris is fairly low, the difficulty in diagnosis, lack of awareness, problematical treatment of BAE, and the resulting fatal consequences highlights that this infection is of great concern, not just for humans but also for animals.[45] Numerous questions about this organism and the resulting disease: BAE remains unanswered. Furthermore, this suggests that a vast number of BAE infections have most likely been unnoticed, and the actual number of BAE cases is significantly higher. For example, there is not a single report of BAE in Africa, despite millions of HIV-infected individuals, who are susceptible hosts to opportunistic pathogens, as well as the warm climate, probable frequent environmental exposure and subordinate sanitation. Previous reports have accounted for approximately 0.1% of total encephalitis cases in otherwise healthy individuals to be caused by Naegleria fowleri or B. mandrillaris to be underestimated (Centers for Disease Control, Prevention; 2008). Consequently, BAE cases should be deliberated as possible causes of encephalitis when patients exhibit general nonspecific encephalitis symptoms. More worryingly, B. mandrillaris has been shown to produce BAE in immunocompetent hosts and so presents a real threat to human and animal health. Current methods of treatment require increased awareness of physicians and pathologists of BAE and strong suspicion based on clinical findings. Early diagnosis followed by aggressive treatment using a mixture of drugs is crucial, and even then the prognosis remains extremely poor. Accordingly, there is an imperative need for the understanding of the pathogenesis and pathophysiology of BAE both at the molecular, cellular, and clinical level as well as the ability of B. mandrillaris to transmit to susceptible hosts, adapt to diverse host and environmental conditions, their ability to overcome host defense barriers and emerge as infective trophozoites to produce CNS infection will provide targets for therapeutic interventions and to design strategies for preventative measures.