Infections with free-living amebae

Handbook of Clinical Neurology, Vol. 114 (3rd series) Neuroparasitology and Tropical Neurology H.H. Garcia, H.B. Tanowitz, and O.H. Del Brutto, Editors © 2013 Elsevier B.V. All rights reserved

Chapter 10

Infections with free-living amebae GOVINDA S. VISVESVARA* Division of Foodborne, Waterborne & Environmental Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA

INTRODUCTION

HISTORICAL PERSPECTIVE

Of the more than 100 species of free-living amebae (FLA) several species belonging to the genus Acanthamoeba, the only known species of Balamuthia, B. mandrillaris, and only one species of Naegleria, N. fowleri, are known to cause central nervous system (CNS) infections in humans and other animals. Acanthamoeba spp. and B. mandrillaris cause an insidious and chronic granulomatous CNS infection known as granulomatous amebic encephalitis (GAE) lasting from a few weeks to 2 years. GAE caused by both Acanthamoeba spp. and B. mandrillaris often disseminates to skin, lungs, kidney, thyroid, and liver. Cases of GAE occur at any time of the year without relation to seasonality. Additionally, Acanthamoeba spp. also cause a sightthreatening infection of the cornea, Acanthamoeba keratitis. Naegleria fowleri, on the other hand, causes an acute and fulminating infection called primary amebic meningoencephalitis (PAM), principally in children and young adults who have had contact with fresh water a few days preceding the infection. PAM usually occurs during summer months when the ambient temperature is high and almost always leads to death within a week to 10 days (Martinez, 1985; Martinez and Visvesvara, 1997; Marciano-Cabral and Cabral, 2003; Schuster and Visvesvara, 2004; Khan, 2006; Visvesvara and Maguire, 2006; Visvesvara et al., 2007b). In addition to these three amebae, a single case of amebic encephalitis caused by Sappinia pedata (previously identified as Sappinia diploidea) has also been described (Gelman et al., 2001; Qvarnstrom et al., 2009). Members of the genus Sappinia have been known to occur in soil contaminated with the feces of lizards, elk, and bison (Page, 1988).

The concept that Acanthamoeba may cause human disease was developed by C. G. Culbertson in 1958 (Culbertson et al., 1958) who isolated an ameba that occurred as a contaminant in the control monkey kidney cell cultures during the development of a vaccine for polio virus. This ameba, now designated as Acanthamoeba culbertsoni, upon inoculation into corticosteroid-treated mice and monkeys, caused encephalitic syndrome in the experimental animals resulting in death. Culbertson hypothesized that Acanthamoeba might also infect humans. The FLA are mitochondria-bearing aerobic organisms that can complete their respective life cycles in the environment without a host in contrast to the parasitic Entamoeba histolytica, which is a mitochondria-lacking anaerobic ameba. Because the FLA live normally as free-living organisms but only occasionally infect humans and other animals and are able to survive within the tissues, they are also called amphizoic amebae. Although quite a few cases of FLA-caused CNS infections have been described the FLA have received little attention because of lack of suspicion and familiarity with these amebae by clinicians and pathologists. Since these infections, especially those caused by Acanthamoeba spp. and Balamuthia, occur often in immunocompromised persons greater numbers of cases would be expected in the developing countries, especially in sub-Saharan Africa and South Asia where the HIV epidemic is flourishing. Because of (a) limited diagnostic expertise in many of these countries, (b) lack of adequate resources for diagnosis, and (c) low rates of autopsy, estimates of the true incidence of these infections are lacking (Visvesvara and Maguire, 2006).

*Correspondence to: Govinda S. Visvesvara, Ph.D., Centers for Disease Control and Prevention, Mailstop D-66, National Center for Emerging and Zoonotic Infectious Diseases, Division of Foodborne, Waterborne & Environmental Diseases, Waterborne Disease Prevention Branch, Roybal Campus, 1600 Clifton Road, Atlanta, GA 30333, USA. Tel: þ1-404-718-4159 (office); þ1-404-718-4174 (main lab), Fax: þ1-404-718-4197, E-mail: [email protected]

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TAXONOMYAND CLASSIFICATION According to the classical taxonomic classification, Acanthamoeba, Balamuthia Sappinia and Hartmannella were classified under Phylum Protozoa, sub phylum Sarcodina, Super Class Rhizopodea, Class Lobosea, Order Amoebida whereas; Naegleria and other Vahlkampfiid amebae were classified under Order Schizopyrenida; Family Vahlkampfiidae (Page, 1988). Recently, the International Society of Protistologists abandoned the older hierarchical systems and replaced it with a new classification based on modern morphological approaches, biochemical pathways, and molecular phylogenetics. According to this new schema the Eukaryotes have been classified into six clusters or “Super Groups” namely, Amoebozoa, Opisthokonta, Rhizaria, Archaeplastida, Chromalveolata, and Excavata. Acanthamoeba and Balamuthia are included under Super Group Amoebozoa: Acanthamoebidae; Naegleria fowleri under Super Group Excavata: Heterolobosia: Vahlkampfiidae; and Sappinia under Super Group Amoebozoa: Flabellinea: Thecamoebidae (Adl et al., 2005).

ACANTHAMOEBA The genus Acanthamoeba was created by Volkonsky in 1931. This genus now includes the ameba that was first isolated from dust in 1913 by Puschkarew and named as Amoeba polyphagus (Page, 1967). Page (Page, 1967) redescribed A. polyphagus as Acanthamoeba polyphaga (Puschkarew). Based purely on morphological criteria, as many as 24 species have been included in the genus Acanthamoeba. These species have been classified under three different groups (Groups 1, 2, and 3) based on differences in the morphology and size of the trophozoites and cysts. Group 1 includes those species that are large amebas with cysts that range in size from 16 to 30 mm (e.g., A. astronyxis, A. comandoni, A. tubiashi, A. echinulata). Group 2 includes by far the largest numbers of species with cysts measuring around 18 mm or less (e.g., A. castellanii, A. polyphaga, A. rhysodes, A. hatchetti). Group 3 consists of species with subtle differences in cysts measuring 18 mm or less (e.g., A. culbertsoni, A. royreba, A. lenticulata) (Pussard, 1964). Because of variation of cyst morphology due to culture conditions species identification based on morphology was considered unreliable and nonmorphological methods were considered necessary to name different species. Currently, sequencing of the 18S rDNA is being used to differentiate isolates and understand the phylogeny of Acanthamoeba. Based on sequence differences 17 genotypes (T1 to T17) of Acanthamoeba have been established (Stothard et al., 1998; Marciano-Cabral and Cabral, 2003; Schuster and Visvesvara, 2004; Khan, 2006;

Visvesvara et al., 2007b; Nuprasert et al., 2010). Several species of Acanthamoeba (A. castellanii, A. culbertsoni, A. polyphaga, A. rhysodes, A. divionensis, A. hatchetti, A. healyi, and A. lenticulata) have been described as causal agents of GAE and skin infections (Martinez, 1985; Martinez and Visvesvara, 1997; Marciano-Cabral and Cabral, 2003; Schuster and Visvesvara, 2004; Khan, 2006; Visvesvara and Maguire, 2006; Visvesvara, 2007; Visvesvara et al., 2007b). The habitat of Acanthamoeba is diverse and has been isolated from soil, dust in the air, fresh, brackish and salt water, Antarctic soil, and ocean dump sites for sewage sludge and acid wastes. They have also been isolated from kitchen and bathroom drains and faucets, home humidifiers, filters of heating, ventilating and air conditioning units, cooling towers, home aquaria, hospital hydrotherapy pools, dental irrigation systems, eye wash stations, bacterial, fungal and mammalian cell cultures, contact lens paraphernalia, ear discharge, pulmonary secretions, swabs obtained from nasopharyngeal mucosa of patients with respiratory complaints as well as healthy individuals, maxillary sinus, mandibular autografts, and stool samples. Additionally, several species of Acanthamoeba have been isolated from tissues of reptiles, birds, and mammals as well as human tissue including brain, lungs, skin, and cornea (Martinez, 1985; Martinez and Visvesvara, 1997; Marciano-Cabral and Cabral, 2003; Schuster and Visvesvara, 2004; Khan, 2006; Visvesvara and Maguire, 2006; Visvesvara, 2007; Visvesvara et al., 2007b). Some of these acanthamoebae have the ability to grow at higher temperatures. For example, a T4 genotype Acanthamoeba that grew at 44 C was isolated from the liver of a toucan (Visvesvara et al., 2007a). It has been shown that many human-infecting Acanthamoeba strains belong to the T4 genotype indicating that some of the animal-infecting and high-temperature-tolerant acanthamoebae may already have developed the ability to infect humans. Acanthamoeba has two stages, a trophic or feeding and dividing stage and a resistant cyst stage, in its life cycle. The trophozoites of Acanthamoeba, depending upon the species and the group to which they are assigned, measure from 14 to 40 mm whereas the cyst measures from 10 to 25 mm in diameter. Both the trophozoite and the cyst possess usually a single nucleus which contains a large, densely staining nucleolus. The trophozoite feeds on bacteria and divides by mitosis. The trophic ameba is characterized by the presence of thorn-like surface projections called acanthopodia. The trophozoite converts into a double-walled cyst when conditions become adverse. The endocyst or the inner cyst wall is usually thick, stellate, polygonal, round, or oval and consists of cellulose  whereas the outer wall (ectocyst) is usually thin, wrinkled, and made up of protein.

INFECTIONS WITH FREE-LIVING AMEBAE

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discovered in A. polyphaga (Raoult et al., 2004). It is quite likely that endosymbiont-bearing Acanthamoeba may serve as reservoir for these bacteria, some of which are potential pathogens for humans signifying its public health importance.

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C Fig. 10.1. (A) Acanthamoeba castellanii trophozoites from a axenic culture, phase contrast, X 600. (B) A. castellanii cysts. Note the wrinkled ectocyst and polygonal endocyst, X 600. (C) Acanthamoeba trophozoites (arrows) in a skin biopsy section stained with hematoxylin and eosin (H&E). X 400.

Pores or ostioles occur at the junction of the ectoand endocysts. The pores are sealed with mucoid plugs (opercula) (Fig. 10.1A, B). Upon favorable conditions the trophozoites emerge from the cysts via the pores (Page, 1967). Recently, Acanthamoeba has generated considerable interest and have been described as “Trojan horses” because of their ability to act as hosts for a wide variety of pathogenic bacteria including Legionella spp., Mycobacterium avium, Listeria monocytogenes, Vibrio cholerae, Francisella tularensis, Burkholderia spp., Helicobacter pylori, Afipia felis, and Escherichia coli serotype O157 (Greub and Raoult, 2004; Berger et al., 2006). According to one report approximately 20 to 24% of clinical and environmental isolates of Acanthamoeba harbor intracellular bacteria (Fritsche et al., 2000). Further, Acanthamoeba infected with Legionella-like bacteria has been isolated from soil samples (Newsome et al., 1998). Additionally, pure cultures of A. polyphaga have been used to isolate L. pneumophila (Rowbotham, 1998), L. anisa (La Scola et al., 2001), and Mycobacterium massiliense (Adekambi et al., 2004) from human clinical specimens such as sputum, liver and lung abscesses, and even human feces. Obligate intracellular pathogens such as Chlamydia, Chlamydophila, and Chlamydia-like bacteria have been found in 5% of Acanthamoeba isolates (Fritsche et al., 2000). Recently, a virus (mimivirus) about the size of a small bacterium with a 1.2-megabase genome has been

Cultivation Since Acanthamoeba spp. are bacterivores they can be easily cultured in the laboratory on non-nutrient agar plates coated with bacteria such as Escherichia coli or Enterobacter aerogenes. The amebae will feed on bacteria, multiply, and cover the surface of the plates within a few days. They will encyst when most of the bacteria have been consumed. Acanthamoeba has also been readily established in bacteria-free or axenic media. Further, many strains of Acanthamoeba have been cultured in chemically defined medium. Additionally, many isolates of Acanthamoeba, especially those isolated from human or animal tissues, have been cultured on mammalian cell cultures (Schuster, 2002).

Granulomatous amebic encephalitis (GAE) GAE caused by Acanthamoeba spp. usually occurs in chronically ill, debilitated individuals, in immunosuppressed patients including those who have HIV/AIDS, or in those who have received broad-spectrum antibiotics or chemotherapeutic medications. Clinical signs include personality changes, headache, low-grade fever, nausea, vomiting, lethargy, diplopia, hemiparesis, seizures, depressed levels of consciousness, and coma. Third and sixth cranial nerve palsies may be seen in some patients. GAE may mimic bacterial leptomeningitis, tuberculous or viral meningitis. Pneumonitis may also occur. Facial palsy resulting in facial asymmetry may also occur. Finally, hemiparesis, seizures, and coma leading to death ensues (Martinez, 1985; Martinez and Visvesvara, 1997; Marciano-Cabral and Cabral, 2003; Schuster and Visvesvara, 2004; Khan, 2006; Visvesvara and Maguire, 2006; Visvesvara, 2007; Visvesvara et al., 2007b). Cases of Acanthamoeba GAE may occur at any time of the year and has no relation to fresh water exposure.

Diagnostic methods CSF in GAE cases often has normal glucose levels whereas protein may be mildly elevated. Lymphocytic pleocytosis is usually seen. Single or multiple heterogeneous, hypodense, non-enhancing, space-occupying lesions involving the basal ganglia, cerebral cortex, subcortical white matter, cerebellum, and pons may be seen, suggesting a brain abscess, brain tumor, or intracerebral hematoma. Computerized tomography (CT) scans of the brain show large, low-density abnormality mimicking a

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single or multiple space-occupying mass. Magnetic resonance imaging (MRI) with enhancements shows multiple, ring-enhancing lesions in the brain (Schumacher et al., 1995; Shirwadkar et al., 2006). Acanthamoeba is generally not found in the CSF, although several case reports have described culturing of amebae from CSF of patients with GAE (Schuster and Visvesvara, 2004). According to one recent report Acanthamoeba was detected in the CSF of a patient without CNS disease and it was believed that the ameba entered from the nasopharynx via a fistula (Petry et al., 2006). Brain and skin biopsies are important diagnostic procedures. Molecular techniques such as PCR and real-time PCR have also been used recently to identify Acanthamoeba in the CSF, brain, skin, and corneal tissue as well as in tear fluid. Acanthamoeba DNA has also been found in the CSF (Bloch and Schuster, 2005) even though amebae were not found. Acanthamoeba spp. can be easily cultured from the brain, skin, lung, and corneal tissue by placing minced tissue on bacterized agar plates. Since the infection becomes apparent only after several weeks or even months the portal of entry is not clearly known. Since Acanthamoeba has been isolated from the nasal passages of humans it is believed that it gains access to humans via airborne dust. According to some studies the presence of Acanthamoeba in human nasal sinuses is in the range of 2% to 24% (Martinez, 1985; Martinez and Visvesvara, 1997; Marciano-Cabral and Cabral, 2003; Schuster and Visvesvara, 2004; Khan, 2006; Visvesvara and Maguire, 2006; Visvesvara, 2007; Visvesvara et al., 2007b). Additionally, it is known that HIV/AIDS patients have sinusitis and ulcers/abscesses in the nasal passages. Amebae may also enter the body through breaks in the skin, resulting in hematogenous dissemination to the lungs and brain (Schuster and Visvesvara, 2004; Visvesvara and Maguire, 2006). A brain or skin biopsy may demonstrate amebic trophozoites and cysts (Fig. 10.1C). Since acanthamoebae are ubiquitous in nature, it is likely that humans may develop antibodies to them. According to some previous studies antibodies to Acanthamoeba have been found in sera of healthy soldiers as well as hospitalized patients in the former Czechoslovakia, adults and children from New Zealand, and patients hospitalized for respiratory problems (Martinez, 1985; Martinez and Visvesvara, 1997; Marciano-Cabral and Cabral, 2003; Schuster and Visvesvara, 2004; Khan, 2006; Visvesvara and Maguire, 2006; Visvesvara, 2007; Visvesvara et al., 2007b). According to another limited study Hispanics were 14.5 times less likely to develop antibodies to Acanthamoeba, especially A. polyphaga, than Caucasians (Chappell et al., 2001). Acanthamoeba spp. also cause infections of the CNS of animals including gorillas, monkeys, dogs, sheep,

cattle, horses, and kangaroos as well as birds, reptiles, amphibians, fishes, and even invertebrates (Martinez and Visvesvara, 1997; Schuster and Visvesvara, 2004; Visvesvara and Maguire, 2006; Visvesvara, 2007; Visvesvara et al., 2007b).

Pathophysiology of Acanthamoeba infections Microscopic examination of CNS sections obtained at autopsy of GAE cases reveals edema, encephalomalachia, and multiple necrotic and hemorrhagic areas. The brainstem, cerebral hemispheres, and cerebellum may show areas of hemorrhagic infarcts. Multinucleated giant cells in the cerebral hemispheres, brainstem, midbrain, cerebellum, and basal ganglion are commonly seen. Occasionally, angiitis may be seen with perivascular cuffing by inflammatory cells, Reactive macrophages are often seen and are mistaken for amebae. Blood vessels are cuffed by polymorphonuclear leukocytes, amebic trophozoites, and cysts (Martinez, 1985; Martinez and Visvesvara, 1997). Since Balamuthia also produces cysts in the brain tissue, either immunochemical tests (Martinez, 1985; Martinez and Visvesvara, 1997; Marciano-Cabral and Cabral, 2003; Schuster and Visvesvara, 2004; Khan, 2006; Visvesvara and Maguire, 2006; Guarner et al., 2007; Visvesvara, 2007; Visvesvara et al., 2007b) or PCR (Booton et al., 2005; Qvarnstrom et al., 2006; Yagi et al., 2008) are needed for diagnosing the infection. Immunodeficient patients may not develop granulomatous reaction but it is commonly seen in immunocompetent patients. Many immunodeficient patients, especially those with HIV/AIDS, develop skin lesions or abscesses or erythematous nodules on the body and limbs (Martinez, 1985; Martinez and Visvesvara, 1997; Seijo Martinez et al., 2000; Marciano-Cabral and Cabral, 2003; Schuster and Visvesvara, 2004; Khan, 2006; Visvesvara and Maguire, 2006; Visvesvara, 2007; Visvesvara et al., 2007b). These nodules are usually firm and nontender but sometimes they become ulcerated and purulent. Acanthamoeba trophozoites and cysts with single nucleus containing a prominent nucleolus are found in pulmonary parenchyma, and skin lesions. In a few cases dissemination to the CNS may not occur (Slater et al., 1994; Walia et al., 2007).

Mechanisms of pathogenesis Several different pathogenic mechanisms are presumably involved during the course of invasion of the tissue. For example, it has been shown by in vitro and experimental in vivo studies that (a) Acanthamoeba produces food cups or amebostomes which help in nibbling away bits and pieces of tissue surface (Pettit et al., 1996; Omana-Molina et al., 2004); (b) Acanthamoeba produces enzymes such as phospholipases, serine proteases, and metalloproteinases as well as plasminogen activators. It

INFECTIONS WITH FREE-LIVING AMEBAE is likely that all of these play a role in the invasion of tissues (Martinez, 1985; Martinez and Visvesvara, 1997; Marciano-Cabral and Cabral, 2003; Schuster and Visvesvara, 2004; Khan, 2006; Sissons et al., 2006; Visvesvara and Maguire, 2006; Visvesvara, 2007; Visvesvara et al., 2007b). Based on in vitro studies it has been shown that the host mounts a defense response in producing interleukins (IL-1a and b, and tumor necrosis factor), which together with macrophage play a role in the killing of amebae (Martinez, 1985; Martinez and Visvesvara, 1997; Marciano-Cabral and Cabral, 2003; Schuster and Visvesvara, 2004; Khan, 2006; Sissons et al., 2006; Visvesvara and Maguire, 2006; Visvesvara, 2007; Visvesvara et al., 2007b).

Therapy and prognosis CNS infections caused by Acanthamoeba are difficult to treat because of several factors. For example (a) the symptoms are nonspecific and can be easily mistaken for other bacterial, viral diseases; (b) a reliable diagnostic test is unavailable; (c) the clinicians who treat the patients are not familiar with FLA and hence diagnosis is made only at autopsy. However, in some cases where a diagnosis was made antemortem by biopsy, especially in cases where a mucocutaneous infection has occurred, treatment with a combination of antimicrobials that included pentamidine, fluconazole, 5 flu cytosine, and sulfadiazine has resulted in success and the patients have survived. Several cases of Acanthamoeba cutaneous infection without CNS involvement have responded well after topical applications of chlorhexidine gluconate and ketoconazole cream in addition to the combinations of drugs as above resulted in therapeutic success (Martinez, 1985; Martinez and Visvesvara, 1997; Marciano-Cabral and Cabral, 2003; Schuster and Visvesvara, 2004; Khan, 2006; Visvesvara and Maguire, 2006; Visvesvara, 2007; Visvesvara et al., 2007b). Additionally voriconazole, a triazole compound, was used on a lung transplant patient with cutaneous acanthamebiasis without CNS involvement (Slater et al., 1994; Walia et al., 2007). Another drug, miltefosine, a hexadecylphosphocholine, has been successfully used to treat a GAE patient in Austria (Aichelburg et al., 2008). In many cases, however, therapy had to be discontinued because of undesirable side-effects of the medications (Martinez, 1985; Martinez and Visvesvara, 1997; Marciano-Cabral and Cabral, 2003; Schuster and Visvesvara, 2004; Khan, 2006; Visvesvara, and Maguire 2006; Visvesvara, 2007; Visvesvara et al., 2007b).

Acanthamoeba keratitis (AK) AK is a painful, vision-threatening infection of the cornea and has occurred primarily in immunocompetent individuals. It is characterized by inflammation of the cornea, severe ocular pain and photophobia, a

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characteristic 360 or paracentral stromal ring infiltrate, recurrent breakdown of corneal epithelium with a waxing and waning clinical course, and a corneal lesion refractory to the commonly used antibacterial, antifungal, and antiviral medications. If proper treatment is not provided, AK may lead to a vascularized scar within a thin cornea, causing impaired vision or perforation of the cornea and loss of the eye. AK is often confused with viral or fungal keratitis and is often misdiagnosed as dendritic keratitis due to herpes simplex virus thus resulting in delay in initiating appropriate therapy to eliminate the amebae (Jones et al., 1975; Stehr-Green et al., 1989; Seal, 2003). The first case of AK in the USA occurred in 1973 in a South Texas farmer, a noncontact lens wearer, who developed ocular trauma of the right eye (Jones et al., 1975). The numbers of cases increased gradually between 1973 and 1984. However, a dramatic increase in AK occurred in 1985, mostly in persons wearing contact lenses. A casecontrol study revealed that a major risk factor was the use of contact lenses and the use of nonsterile home-made saline solution (Stehr-Green et al., 1989; Visvesvara et al., 1990). Because of a multistate outbreak of Acanthamoeba keratitis during 20062007, an epidemiological survey conducted by Centers for Disease Control and Prevention (CDC) revealed that the national increase in the number of AK cases was associated with the use of Advanced Medical Optics Complete® MoisturePlus™ multipurpose contact lens solution, leading to an international recall by the manufacturer (Joslin et al., 2006; Verani et al., 2009).

PATHOPHYSIOLOGY OF AK Once the amebae enters the eye Acanthamoeba will adhere to the corneal epithelial cells and secrete proteolytic and collagenolytic enzymes that may damage the corneal epithelium and thus contribute to the pathogenesis of AK. According to recent studies, the initial process of invasion occurs when a 136-kDa mannosebinding protein (MBP), a lectin, expressed on the surface of Acanthamoeba, adheres to mannose glycoproteins on the surface of the epithelial cells and destroys the epithelial cells. Acanthamoeba may also produce food cups on its surface and ingest the epithelial cells (Clarke and Niederkorn, 2006; Garate et al., 2006a, b).

DIAGNOSTIC MICROBIOLOGY AK is usually diagnosed by performing culture of corneal scraping and or biopsy and staining (Giemsa-Wright or trichrome) of the corneal smears. Recently, however, confocal microscopy has been used in diagnosis of AK (Joslin et al., 2006; Parmar et al., 2006). Molecular techniques such as PCR and real-time PCR have also been

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used to identify Acanthamoeba in the corneal tissue as well as tear fluid (Qvarnstrom et al., 2006; Riviere et al., 2006).

MANAGEMENT AK has often been treated successfully by applying either polyhexamethylene biguanide (PHMB) or chlorhexidine gluconate with or without Brolene (Seal, 2003; Clarke and Niederkorn, 2006). When medical treatment failed, a combination of debridement and penetrating keratoplasty have been used with good results in some cases

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PREVENTION Since contact lenses and lens-care solutions are wellknown risk factors for AK, eye care professionals need to educate patients about the proper care and use of contact lenses. Contact lenses and contact lens paraphernalia, particularly the solutions, should be kept meticulously clean. Contact lens wearers should follow the directions and recommendations of the manufacturers and eye care professionals. Additionally, patients should be instructed not to wear contact lenses during swimming, performing water-sport activities, or relaxing in a hot tub or Jacuzzi. As Acanthamoeba spp. can grow and colonize hot water tanks, jacuzzis, filters used in heating, ventilating and air conditioning units, and in-line filters used for purifying portable water supplies and eye wash stations, periodic inspection of these systems is recommended.

BALAMUTHIA MANDRILLARIS Balamuthia mandrillaris was isolated in 1986 from an ulcer in the cerebrum of a pregnant mandrill baboon that died of GAE in the san Diego Animal Park. It was initially described as a leptomyxid ameba because of the resemblance of the cysts to those of a soil ameba included under the family Leptomyxidae. In 1993 it was recognized as being different from the leptomyxids and was designated as a new genus and species, Balamuthia mandrillaris (Visvesvara et al., 1990, 1993). It is the only species included in the genus Balamuthia. Infection caused by this ameba is similar to GAE caused by Acanthamoeba and can occur at any time of the year and is not dependent upon a particular season. Balamuthia causes an insidious, chronic, and subacute GAE in both immunocompromised and immunocompetent individuals that may last anywhere from several weeks to 2 years (Visvesvara et al., 1993; Schuster and Visvesvara, 2004; Visvesvara et al., 2007b). Recently, however, two clusters of acute infection due to Balamuthia occurred in recipients of solid organ transplantation resulting in

C Fig. 10.2. (A) Balamuthia mandrillaris trophozoite from culture, phase contrast, X 600. (B) B. mandrillaris cyst, X 600. (C) B. mandrillaris trophozoite (arrow) in a brain section stained with H&E, X 400.

the death of several patients within 3 weeks (CDC, 2010a, b). Balamuthia mandrillaris, like Acanthamoeba, has only two stages in its life cycle (Fig. 10.2A, B). The trophozoites are larger than those of Acanthamoeba, pleomorphic, and measure from 12 to 60 mm with a mean of about 30 mm. The trophozoites usually are uninucleate, but binucleate forms are occasionally seen. The nucleus normally has a single, large, centrally placed, dense nucleolus. Occasionally, however, they may possess, especially in the infected brain tissue, two or three nucleoli (arrow in Fig. 10.2C). Cysts also have a single nucleus, and range in size from 12 to 30 mm with a mean of 15 mm. Under the light microscope, cysts appear to be double walled, with a wavy outer wall and a round inner wall. Ultrastructurally, however, the cysts possess three walls  an outer thin and irregular ectocyst, an inner thick endocyst, and a middle amorphous fibrillar mesocyst (Visvesvara et al., 1990, 1993).

Cultivation Balamuthia mandrillaris will not grow on bacteriacoated agar plates (Berger et al., 2006). Although the exact nutritional requirement of this ameba has not been

INFECTIONS WITH FREE-LIVING AMEBAE determined it is believed that it feeds on small amebae and fungi in its environmental niche (Schuster and Visvesvara, 2004). In the laboratory Balamuthia can be grown on mammalian cell cultures such as monkey kidney (E6), human lung fibroblasts (HLF), and human brain microvascular endothelial cells. Balamuthia can also be grown axenically in a complex cell-free chemical medium containing fetal bovine serum (Schuster, 2002).

Diagnosis: clinical and laboratory methods Balamuthia mandrillaris is not readily isolated from the CSF, although in two cases it was isolated from the CSF obtained at autopsy (Jayasekera et al., 2004; CDC, 2010a). Microscopic examination of the CSF reveals normal to low glucose but increased protein levels and lymphocytic pleocytosis with less than 500 cells/mm3. Balamuthia mandrillaris has been cultured antemortem from brain biopsies from several patients. In a majority of cases, however, final diagnosis was made only at autopsy. It is difficult to differentiate B. mandrillaris from Acanthamoeba spp. in formalin-fixed and paraffin-embedded tissue sections by light microscopy because of similar appearance. However, they can be differentiated by immunohistochemical techniques by using rabbit anti-Acanthamoeba or anti-B. mandrillaris sera (Visvesvara et al., 1990, 1993; Martinez and Visvesvara, 1997; Booton et al., 2003; Visvesvara, 2007; Visvesvara et al., 2007b). They can also be differentiated by transmission electron microscopy because the cysts of Balamuthia have three layers in contrast to Acanthamoeba which has two cyst walls. In vitro cultures of Balamuthia can be established by inoculating fresh or frozen brain and or skin tissue into mammalian cell culture (Schuster, 2002; Schuster and Visvesvara, 2004; Visvesvara et al., 2007b; CDC, 2010a, b).

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to coma and death. MRI may show one or more low-density lesions initially but as the infection progresses the lesions may increase in size. CT and MRI scans may indicate hemorrhage within lesions and may show up as “space-occupying lesions” mimicking a brain abscess, brain tumor, or intracerebral hematoma. Some patients have also been erroneously diagnosed as neurotuberculosis or neurocysticercosis (Martinez and Visvesvara, 1997; Schuster and Visvesvara, 2004; Visvesvara, 2007; Visvesvara et al., 2007b). The brain is edematous and has uncal and tonsillar herniation. Inflammation is seen in the white matter in the brainstem, cerebral hemispheres, and cerebellum. The necrotic areas are filled with neutrophils, mononuclear cells, and multinucleated giant cells. Balamuthia trophozoites and cysts are seen interspersed with the neutrophilic infiltrates and macrophages. The trophozoite nucleus sometimes contains more than one nucleolus, a feature characteristic of Balamuthia (Fig 10.2C). The blood vessel walls are also infiltrated with amebae (Martinez and Visvesvara, 1997; Bravo et al., 2011).

Mechanisms of pathogenesis Balamuthia is known to ingest bits and pieces of host tissue as well as producing enzymes that degrade the tissue. Recently it has been shown that Balamuthia induces human brain microvascular endothelial cells to release a pleiotropic cytokine, IL-6, which is known to play a role in initiating early inflammatory response (Matin et al., 2006). Further, it has been shown that B. mandrillaris interacts with the host connective tissue containing collagen-1, fibronectin, and laminin-1, which is probably related to the production of food cups thus enhancing the pathological process (Rocha-Azevedo et al., 2007).

Pathophysiology of Balamuthia infections

Molecular characterization

The incubation period is not known except in transplanttransmitted cases (see above) and the course of infection may last as long as several weeks to 2 years. Many cases develop cutaneous lesions or ulcers prior to CNS infection. These skin lesions may occur on the face, trunk, and/or extremities (Bravo et al., 2006; Bravo et al., 2011). Balamuthia cases have occurred in children and older individuals with no known immunodeficiency in addition to patients with HIV/AIDS and other immunodeficient patients, diabetic patients, and those undergoing therapy with corticosteroids (Martinez and Visvesvara, 1997; Schuster and Visvesvara, 2004; Visvesvara and Maguire, 2006; Visvesvara, 2007; Visvesvara et al., 2007b). Symptoms may initially include headache, meningismus, nausea and vomiting, low-grade fever, and lethargy. Later on visual disturbances, facial nerve palsy, ataxia, seizures, and hemiparesis may develop leading

Molecular techniques have been developed to identify Balamuthia DNA in the CSF, brain tissue, and even in the environment (Booton et al., 2003; Ahmad et al., 2011). Specific primers have been developed to retrospectively confirm Balamuthia infections in archived slide specimens fixed in formalin and embedded paraffin (Tavares et al., 2006; Yagi et al., 2008). Additionally, a real-time multiplex PCR assay has been developed that can specifically identify Balamuthia DNA in human CSF and brain tissue in about 4 to 5 hours (Qvarnstrom et al., 2006).

Balamuthia antigens and antibodies A recent study using SDS-PAGE and Western blot techniques has shown that eight isolates of B. mandrillaris, irrespective of human or animal or geographical origin,

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despite exhibiting some minor differences, were all basically similar thus corroborating previous studies based on molecular sequencing (Kucerova et al., 2011). According to some recent studies antibodies to B. mandrillaris have been identified in healthy persons without any symptoms and in Balamuthia-infected patients. It is believed that the acquisition of such antibodies is probably due to exposure to the Balamuthia present in the environment as B. mandrillaris has been either isolated from or identified in the environment (Schuster et al., 2006b). Huang et al., using flow cytometry, detected high titers of anti-Balamuthia antibodies in healthy adults, children, and even in cord blood from South Australia (Huang et al., 1999). Schuster et al. using an immunofluorescence test (Schuster et al., 2006b; Schuster et al., 2009) detected high titer antibodies to Balamuthia only in patients hospitalized with Balamuthia encephalitis. Further, Schuster et al. developed an ELISA test and subsequently confirmed that the patients with Balamuthia encephalitis did develop high titer antibodies to this organism (Schuster et al., 2008). Recently, Kiderlen et al., using a flow cytometry-based assay, tested sera obtained from German blood donors, West African rain forest workers, and patients with atypical encephalitis, pneumonitis, amebiasis, and toxoplasmosis. They found 92% of persons involved in a primate project in West Africa had high titers of antibodies. Of these, 15 (50%) had very high titers, comparable to sera from proven cases of balamuthiasis. Sera collected from European donors living in West Africa had low titers. But the sera collected from West Africans belonging to traditional farming and hunting communities had very high titers. Interestingly, the older these West Africans the greater were the anti- Balamuthia titers. Of the nine individuals with the highest titers, most were elderly men professing intensive outdoor activity (Kiderlen et al., 2009). According to Kiderlen et al. (Kiderlen et al., 2009), these West Africans may have had constant contact with some other amebae that were antigenically related to B. mandrillaris and therefore developed high antibody titers to B. mandrillaris. These authors concluded that such high titers might have been due to actual infections with Balamuthia that were successfully cleared, indicating that not all infections with B. mandrillaris are lethal. Balamuthia GAE has also been reported in a variety of animals including gorillas, baboons, gibbons, monkeys, horses, sheep, and dogs (Rideout et al., 1997; Schuster and Visvesvara, 2004; Visvesvara et al., 2007b). An animal model using severe combined immunodeficient (SCID) mice has been developed to study Balamuthia GAE (Janitschke et al., 1996). When trophozoites of B. mandrillaris are intranasally instilled into immunodeficient mice they adhere to the nasal

epithelium, migrate along the olfactory nerves, traverse the cribriform plate, and invade the brain, similar to the invasion pathway described for N. fowleri (Kiderlen and Laube, 2004). Additionally, Kiderlen et al. have shown that both immunodeficient and immunocompetent mice can be infected per orally with Balamuthia and that the amebae migrate to the CNS. Balamuthia antigens but not amebae are seen in the fecal pellets of mice (Kiderlen et al., 2007).

Therapy and prognosis Although Balamuthia GAE results in death in most cases several patients have recovered from this terrible infection after treatment with a combination of antimicrobials including pentamidine isethionate, sulfadiazine, clarithromycin, fluconazole, and flucytosine (5-fluorocytosine) (Deetz et al., 2003; Jung et al., 2004; Cary et al., 2010; Doyle et al., 2011). Additionally, albendazole and itraconazole and surgical excision have also been used in the case of two Peruvian patients with cutaneous lesions (Bravo et al., 2006). Miltefosine has been used to treat patients with balamuthiasis and transplant-transmitted Balamuthia infections (CDC, 2010a, b; Martinez et al., 2010). In vitro studies have shown that pentamidine and propamidine isethionate at a concentration of 1 mg/mL inhibit growth of Balamuthia amebae by 82% and 80%, respectively, but are not amebacidal. However, a number of pharmaceuticals including macrolide antibiotics, azole compounds, gramicidin, polymyxin B, trimethoprim, sulfamethoxazole, and a combination of trimethoprimsulfamethoxazole as well as amphotericin B had either very little or no activity (Schuster et al., 2006a). Investigations with newer formulations are urgently needed to identify drugs that would be beneficial in the treatment of this devastating infection.

Epidemiology Epidemiology of Balamuthia GAE is not well understood. However, based on several case reports it can be hypothesized that gardening, playing with dirt, or exposure to dust while motor cycling may play significant roles in the acquisition of Balamuthia infection. It has been shown that Balamuthia is found in the soil and dust in air (Schuster et al., 2003; Dunnebacke et al., 2004; Niyyati et al., 2009) and the route of infection is probably via inhalation of Balamuthia either present in soil or carried by the wind or the amebae entering the body through a cut or puncture. Once it gains entry into the body it is likely to spread hematogenously to the brain. Additionally, there is some indication that water may serve as a vehicle for acquiring the infection since two dogs died of balamuthiasis after swimming in stagnant ponds (Foreman et al., 2004; Finnin et al., 2007).

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It is also possible that some groups of people may be genetically predisposed or may have other undetermined factors for infection with Balamuthia (Schuster et al., 2004). Balamuthia may act as hosts for pathogenic microorganisms such as Legionella (Shadrach et al., 2005).

Prevention and control

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B

C

Cases of balamuthiasis have occurred in hosts with weakened immune systems, children, and older individuals and presently no clearly defined methods are available for the prevention of infection with these amebae.

NAEGLERIA FOWLERI Naegleria fowleri is the only human pathogenic species in the genus Naegleria that contains 47 species (De Jonckheere, 2011). It is a free-living, thermophilic ameboflagellate that causes an acute and fulminating infection of the CNS called primary amebic meningoencephalitis in children and young adults with a history of fresh water contact a couple of weeks prior to the onset of symptoms. PAM almost always results in death (Martinez, 1985; Marciano-Cabral, 1988; Martinez and Visvesvara, 1997; Schuster and Visvesvara, 2004; Visvesvara and Maguire, 2006; Visvesvara, 2007; Visvesvara et al., 2007b). Naegleria fowleri has three stages in its life cycle, a trophozoite, a cyst, and a flagellate; hence it is also called an ameboflagellate. The trophozoite is a slug-like ameba, feeds on gram-negative bacteria and reproduces by binary fission. It exhibits rapid sinusoid movement by producing anteriorly hemispherical bulges, lobopodia. The posterior end, the uroid, is sticky and often has several trailing filaments. The trophozoite differentiates into a pear-shaped biflagellate stage in response to sudden changes in the environment. The trophozoite measures 10–25 mm and has a single round nucleus which contains a large, centrally placed, dense nucleolus. The cytoplasm contains numerous mitochondria, ribosomes, food vacuoles, and a contractile vacuole (Fig. 10.3A). The trophozoite transforms into a flagellate stage when the ionic concentration of the surrounding environment changes but reverts back to the trophic stage during favorable conditions. In the laboratory, this transformation can be induced by washing trophozoites in distilled water. Flagellates will emerge within an hour. The flagellate has a single nucleus with a large nucleolus and usually has two flagella but three or four flagella may also be seen occasionally. The flagellate does not have a cytostome and hence cannot feed. It ranges in length from 10 to 16 mm (Fig. 10. 3B). The trophozoite transforms into the resistant cyst during adverse conditions when food supply becomes scarce or the environmental niche dries. The cyst is usually

D Fig. 10.3. Naegleria fowleri trophozoite (A); flagellate (B); and cyst (C) from culture. Phase contrast. X 1,000. D. N. fowleri trophozoite in a brain section stained with H&E. X 400.

spherical, measures 7 to 14 mm, and is double-walled with a thick endocyst and a closely apposed thinner ectocyst. The cyst wall has pores but may not be clearly seen (Fig. 10.3C). The cyst has a single nucleus with a prominent nucleolus (Marciano-Cabral, 1988; Page, 1988).

Cultivation Naegleria fowleri can be cultured on non-nutrient agar plates coated with Escherichia coli or Enterobacter aerogenes. The amebae will feed on the bacteria, multiply, and differentiate into cysts within a few days. They can be easily subcultured by cutting out a small piece of agar containing trophozoites and/or cysts and placing it face down onto a fresh agar plate coated with bacteria as before. It can also be cultured axenically in a complex chemical medium with 5% fetal bovine serum in the absence of bacteria as well as on mammalian cell cultures (Marciano-Cabral, 1988; Schuster, 2002).

Primary amebic meningoencephalitis PAM occurs in active healthy children and young adults with no immunodeficiency who have had a history of recent contact with fresh water by participating in swimming, diving, or other aquatic activities in man-made bodies of water, or unchlorinated swimming pools. The ameba enters through the nasal passages and cross the cribriform plate and reaches the subarachnoid space and disseminates into the olfactory lobes. The incubation period of PAM varies from 2 to 15 days. Symptoms include severe headache, nausea, vomiting, fever, stiff neck, and behavioral abnormalities. Other symptoms

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may include abnormalities in taste or smell and cerebellar ataxia, photophobia, and an increase in intracranial pressure, lethargy, confusion, generalized seizures, and coma. Death usually occurs within a week to 10 days (Carter, 1972; Martinez, 1985; Marciano-Cabral, 1988; Martinez and Visvesvara, 1997; Schuster and Visvesvara, 2004; Visvesvara and Maguire, 2006; Visvesvara, 2007; Visvesvara et al., 2007b). Because of the absence of distinctive symptoms or clinical features PAM can be mistaken for pyogenic or bacterial meningitis thus resulting in delayed diagnosis. Therefore, most cases have been diagnosed only at autopsy based on the identification of N. fowleri by immunochemical tests or molecular methods (Martinez, 1985; Martinez and Visvesvara, 1997; Schuster and Visvesvara, 2004; Qvarnstrom et al., 2006; Visvesvara and Maguire, 2006; Guarner et al., 2007; Visvesvara, 2007; Visvesvara et al., 2007b). PAM has been described from all over the world. Although the first human case of amebic meningoencephalitis due to N. fowleri was described in Australia in 1962 it was described as due to Acanthamoeba at the time. PAM is not really a new disease as retrospective examination of brain tissue has revealed that it had occurred as far back as 1901 (Dos Santos, 1970; Martinez, 1985; Martinez and Visvesvara, 1997; Schuster and Visvesvara, 2004; Visvesvara and Maguire, 2006; Visvesvara, 2007; Visvesvara et al., 2007b). Naegleria fowleri also causes infection leading to death in animals in zoos and domestic animals, especially cattle (Lozano-Alarcon et al., 1997; Daft et al., 2005).

Diagnostic microbiology The CSF obtained by lumbar puncture appears to be pleocytotic, with an abundance of polymorphonuclear leukocytes. The CSF pressure is usually elevated (100–600 mmHg), with low to normal glucose concentration and high protein content, ranging from 100 mg/ 100 mL to 1000 mg/100 mL. CSF has no bacteria but when examined in situ amebae are often seen but mistaken for leukocytes. CT scans show obliteration of the cisterns around the midbrain and the subarachnoid space. Marked diffuse enhancement in these regions may be seen after administration of intravenous contrast medium (Schumacher et al., 1995). Smears of CSF should be stained with Giemsa or trichrome for detecting the ameba with the characteristic nuclear morphology. Gram stain is not useful. Many cases have been diagnosed retrospectively based on examination of hematoxylin and eosin-stained sections, immunohistochemical tests, and PCR assay. A real-time multiplex PCR assay developed at CDC identifies N. fowleri DNA in the CSF within 3 to 4 hours, thus greatly facilitating a quick diagnosis (Qvarnstrom et al., 2006).

Pathological features At autopsy the cerebral hemispheres are swollen and edematous. The olfactory bulbs and the orbitofrontal cortices are characterized by hemorrhagic necrosis and are surrounded by purulent exudates containing predominantly polymorphonuclear leukocytes (PMN), a few eosinophils, a few macrophages, and some lymphocytes (Dos Santos, 1970; Carter, 1972; Martinez, 1985; Martinez and Visvesvara, 1997). The cerebral hemispheres are usually soft, swollen, edematous, and severely congested. The leptomeninges (arachnoid and pia mater) are severely congested, diffusely hyperemic, and opaque with limited purulent exudate within sulci, base of the brain, brainstem, and cerebellum. Amebic trophozoites, but not cysts, are usually seen within the Virchow–Robin spaces with minimal or no inflammatory reaction. Amebae, ranging in size from 8 to 12 mm and possessing densely staining round nuclei, are found in large numbers in the base of the brain, hypothalamus, and midbrain (Fig. 10.3D). The amebae can be specifically identified as N. fowleri by histochemical techniques using polyclonal or monoclonal antibodies.

Mechanisms of pathogenesis Our current knowledge of the virulence factors of N. fowleri is limited. However, based on in vitro studies it has been shown that N. fowleri when inoculated into tissue culture cells destroy the cell monolayer by causing cytopathic effect (CPE). The amebae produce suckerlike appendages or amebostomes that “nibble” away at the tissue culture cells. Additionally, the amebae produce (a) phospholipase A and B activity or a cytolytic factor causing destruction of cell membranes; (b) neuraminidase or elastase activity facilitating destruction of tissue culture cells; (c) presence of a perforin-like, poreforming protein that lyses target cells; and (d) the presence of a cytopathic protein that triggers the apoptosis pathway in susceptible tissue culture cells (MarcianoCabral, 1988; Schuster and Visvesvara, 2004). Mice can be infected intranasally with N. fowleri trophozoites to produce PAM that resembles the human disease.

Epidemiology Naegleria fowleri occurs worldwide and has been isolated from soil; fresh water; cooling towers of electric and nuclear power plants; heating, ventilating and air conditioning (HVAC) units, hot springs; hydrotherapy and remedial pools; aquaria; sewage; dust in the air and even the nasal passages and throats of healthy individuals. Since N. fowleri is thermophilic and can tolerate temperatures of up to 45  C warm fresh waters coupled

INFECTIONS WITH FREE-LIVING AMEBAE with a bacterial food supply are ideal habitats for these amebae (Marciano-Cabral, 1988; Schuster and Visvesvara, 2004). A study of N. fowleri isolates from the USA and Mexico confirmed the presence of two major groups of N. fowleri strains: the European–American group (genotype 3) and representatives of the Wide spread group (genotype 1). A third group (genotype 2, restricted so far to America only) was also described indicating that some N. fowleri strains are geographically segregated (Zhou et al., 2003). Based on case reports in the literature almost all of the cases had a history of swimming in warm freshwater during summer months when ambient temperature is likely to be high (Visvesvara et al., 2007b; Yoder et al., 2010). PAM caused by N. fowleri has been recorded from around the world including the USA. As of August 2011, as many as 131 cases of PAM have occurred in the USA. PAM is an acute and fulminating disease, developing within several days following exposure to the fresh water source, and causing death within 5 to 10 days after hospitalization. Additionally, 75% of these cases were males of about 12 years of age. A recent review at CDC documented 111 cases of PAM in the USA since 1962 (Yoder et al., 2010). While this organism and PAM cases have historically been found in southern-tier states, warming temperatures that are expected with climate change might provide favorable conditions for growth of this organism in northern latitudes (Yoder et al., 2010). According to the forecast by a United Nations scientific advisory panel, global temperature will rise 0.8  C–3.5  C by the year 2100 if production of greenhouse gases is not reduced. An increase in surface temperature will create ideal niches for the thermophilic N. fowleri (Cogo et al., 2004). Therefore, people who have contact with fresh water as a result of bathing, swimming, diving, or water skiing in pools or freshwater bodies of water will have increased chances of becoming infected with N. fowleri (Yoder et al., 2010).

Immunology An antibody response to N. fowleri has been documented in a patient who recovered from PAM (1, 100) (Seidel et al., 1982; Visvesvara et al., 2007b). The antibody persisted for almost 4 years. Another patient who was kept alive for 3 weeks on a ventilator also developed an antibody response to N. fowleri. Apart from these observations it is believed that since most PAM patients die within a short time (5–10 days), there is insufficient time to mount a detectable antibody response and hence the usefulness of serological tests in the diagnosis of PAM is doubtful. In a previous study, using an agglutination test, it was shown that sera from individuals residing in southeastern states of the USA had significantly greater

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agglutinating ability than those obtained from Pennsylvania, a northeastern state, and the agglutinating antibody is of the IgM class (Marciano-Cabral et al., 1987).

Biochemical and molecular techniques Although isoenzyme analysis of cultured amebae and monoclonal antibodies (MAb) have been used to identify N. fowleri, more precise DNA-based molecular tests such as PCR and nested PCR and real-time PCR assays are being used currently for the specific identification of N. fowleri in the CNS and the environment (Reveiller et al., 2002; Qvarnstrom et al., 2006). Additionally specific genotypes of N. fowleri can be distinguished by sequencing of the 5.8S rRNA gene and the internal transcribed spacers 1 and 2 (ITS1 and ITS2) (De Jonckheere, 2011). Based on the sequencing of the ITS of the clinical isolates it has been shown that the two strains of N. fowleri, isolated from two PAM patients who visited the same hot spring in California but at different times, belonged to the same genotype, type 2 (Zhou et al., 2003). A real-time PCR assay specifically identifies N. fowleri DNA in the CSF and brain tissue samples obtained from PAM patients antemortem. This test is fast (within 3 to 4 hours from the receipt of the specimen), accurate, and identifies all three genotypes known to be present in the USA (Qvarnstrom et al., 2006). This test can be multiplexed so as to identify all three amebae (Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri).

Therapy and prognosis Because PAM is an acute and fulminating infection resulting in death within a short period of time only a few patients have survived. One of these survivors, a Californian girl, was aggressively treated with intravenous and intrathecal amphotericin B, intravenous and intrathecal miconazole, and oral rifampin (Seidel et al., 1982). Since amphotericin B is water insoluble and has renal toxicity other drugs with less or no toxicity are urgently needed. In vitro studies of phenothiazine compounds (chlorpromazine and trifluoperazine), which can accumulate in the central nervous system (CNS), were found to have inhibitory effects on N. fowleri (Schuster and Visvesvara, 2004). Azithromycin, a macrolide antimicrobial, has been shown to be effective against Naegleria both in vitro and in a mouse model of PAM (Goswick and Brenner, 2003). Other in vitro data revealed that N. fowleri was sensitive to the triazole compound voriconazole at low concentrations ( 10 mg/mL) while concentrations  10 mg/mL were amebacidal (Schuster et al., 2006a). A whole genome sequence of N. fowleri that is underway (Herman et al., 2011) would greatly facilitate studies to investigate exact mechanisms

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of pathogenesis as well as provide potential targets for drug discovery.

Prevention Naegleria fowleri is susceptible to chlorine and are killed at one part per million. Based on a retrospective study it has been shown that an inadequately chlorinated swimming pool was the venue for 16 deaths over a 3-year period in the former Czechoslovakia (Martinez, 1985; Schuster and Visvesvara, 2004; De Jonckheere, 2011). Nineteen deaths were traced to unfiltered domestic water piped into homes in Southern Australia. Water was piped overland for long distances through desert and consequently the water was heated. When kids swam and played in pools fed with the warm water they contracted PAM and died. Because of this the South Australia High Commission established the ameba monitoring program which routinely determined residual chlorine levels and the total coliform counts to identify if N. fowleri is present in such waters. They also conducted a public safety campaign to educate the public to minimize the incidence of PAM (Martinez, 1985). Domestic water supply had figured as a source of N. fowleri infection in two children in Arizona who played in a wading pool filled with the domestic water (Okuda et al., 2004). Naegleria fowleri was identified in the water by nested PCR analysis (Marciano-Cabral et al., 2003). This was the first time that domestic water supply was implicated as the source of N. fowleri in the USA. It is therefore important to properly chlorinate domestic water supplies and swimming pools. Many cases of PAM have occurred in people engaged in aquatic activity in natural bodies of water such as lakes, ponds and streams or geothermal pools or hot springs that cannot be chlorinated and where N. fowleri may proliferate.

SAPPINIA PEDATA Gelman et al. (Gelman et al., 2001) reported the first and only case of amebic encephalitis caused by another freeliving ameba identified at that time as Sappinia diploidea based on the nuclear morphology of the ameba. Recently this case was re-identified as S. pedata based on molecular sequencing (Qvarnstrom et al., 2009). This infection occurred in an immunocompetent, previously healthy 38-year-old male. The patient lost consciousness for a short time and developed nausea and vomiting followed by bifrontal headache, photophobia, and blurry vision that lasted 2 to 3 days. MRI showed a solitary 2 cm mass in the posterior left temporal lobe. The excised mass on microscopic examination revealed necrotizing hemorrhagic inflammation containing amebic organisms (Fig. 10.4C). Members of the genus Sappinia are coprozoic amebas

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C Fig. 10.4. (A) Sappinia pedata trophozoite from culture; (B) S. pedata cyst, both at X1,000. (C) S. pedata in a brain section stained with H&E. X 1,000.

found in feces of elk, bison, and perhaps cattle. The life cycle of this ameba includes a feeding trophic and a dormant cyst stage (Fig. 10.4A, B). Both trophozoites and cysts are binucleate, with two tightly apposed nuclei. The trophozoite measures 40 to 80 mm, is ovoid or oblong, and appears to be flattened with occasional wrinkles on the surface. The cytoplasm contains a contractile vacuole and food vacuoles. The mature cyst is round and measures 15 to 30 mm. Both species of Sappinia (S. diploidea and S. pedata) can be cultivated on non-nutrient agar plate coated with bacteria (Schuster, 2002).

CONCLUSIONS Encephalitis caused by the free-living amebae Acanthamoeba, Balamuthia and Naegleria, although rare, is of major concern, because children and young adults are often involved and mortality is high. The high death rate is in large part due to delay in diagnosis because of the unfamiliarity of the caregivers with this infection and the lack of optimal antimicrobial therapy. Most of the cases of amebic encephalitides reported in the literature have been from countries where well-developed medical facilities are available. However, in the developing countries, especially in sub-Saharan Africa, where facilities are either minimal or lacking, many cases might have gone undiagnosed (Visvesvara and Maguire, 2006).

Disclaimer The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

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