Primary amoebic meningoencephalitis (PAM) is an infection of the central nervous system (CNS) caused by a free-living amoeba known as Naegleria fowleri . N. fowleri, a thermophilic amoeboflagellate, is normally found in warm freshwater bodies such as lakes, rivers, hot springs, and other thermally polluted waters[1] . The infection is contracted when contaminated water is forcibly drawn into the nasal cavity. //N. fowleri// adheres to the nasal mucosa, and subsequently migrates to the olfactory bulbs, using the olfactory nerves for guidance[2] . Once inside the brain, N. fowleri proliferates and initiates cytotoxic effects that cause inflammation and necrotic hemorrhaging[3] . Symptoms typically appear within 2-15 days after exposure. The symptoms include headache, fever, stiff neck, vomiting, compromised neurological and mental functions, seizures, and coma. The disease is rapidly progressive and highly fatal. Death typically occurs within 3-7 days after the first sign of symptoms[2] .

Only 235 cases of PAM have been reported worldwide since it was discovered in 1968.[4] Despite its rare occurrence, medical professionals currently face many challenges with the disease. Awareness is low among clinicians, diagnosis is difficult and time consuming, and treatment is unsuccessful in most cases. In addition, millions of people are exposed to the amoeba annually; however, no reliable estimation of occurrence of PAM exists. Current research primarily investigates novel therapies, diagnostic assays, and the underlying pathogenesis of the disease[2] .

Fig. 1. Naegleria fowleri in its three distinct morphological states

1.1 Pathogenesis

1.1a Cause: Naegleria fowleri

Primary amoebic meningoencephalitis (PAM) is a rare and rapidly fatal infection of the human central nervous system (CNS) caused by a free-living protist called Naegleria fowleri. The Naegleria genus belongs to a family collectively known as Vahlkamphfidae[4] . Although more than 40 species of Naegleria have been identified, N. fowleri is the only pathogenic species associated with a human infection[5] . N. fowleri is a thermophilic amoeba capable of surviving and proliferating in temperatures as high as 45 degrees Celsius[6] . Its geographic distribution throughout the world is ubiquitous. N. fowleri has been found in warm freshwater of tropical, subtropical, and temperate environments. It is naturally found in lakes, rivers, ponds, hot springs, puddles, and soil. In addition, it is also found in tap water, baths, swimming pools, sewage canals and in the effluents of power plants because of thermal pollution and poor maintenance. It survives by feeding on bacteria[4] .

1.1b Life Cycle

N. fowleri is a triphasic amoeboflagellate. Its life cycle consists of 3 distinct morphologically and functional states. Its existing state depends on its environment[7] .

Fig. 2. Life cycle of Naegleria fowleri

Cyst Stage
The cyst is the resistant morphological state of N. fowleri that occurs under hostile environmental conditions. Although nonpathogenic in this stage, cysts may also enter the nasal passageway, where they can then convert to trophozoites and induce infection[5,7] .

Trophozoite Stage
The trophozoite is the pathogenic form of N. fowleri for humans. In this state it is capable of proliferation and feeding. Proliferation occurs through binary fission. A 'food-cup' structure, called an amoebastome, develops on the surface of the amoeba for feeding. The elongated, slug-like trophozoite is typically 10-25µm in size and is capable of locomotion by means of pseudopodia
[5,7] .

Flagellate Stage
The flagellate is a transient morphological state of N. fowleri that occurs when deprived of nutrients in water. The characteristic feature of N. fowleri during this stage is a pear-shaped structure. N. fowleri is nonpathogenic in this stage[5,7] .

1.1c Infection

Infection occurs when the trophozoite adheres to the nasal mucosa after contaminated water enters the nasal cavity.
Fig. 3. Electron micrograph of Naegleria fowleri trophozite feeding using amoebastomes.
The adherence of the trophozoite to neurons and other cell types involves binding to extracellular matrix glycoproteins such as fibronectin, collagen, and laminin; at least in vitro[8] [7]. For example, Han et al, identified a fibronectin binding surface protein in extracts of N. fowleri using binding assays[9] . Once adhered to the nasal mucosa, the trophozoite migrates to the olfactory bulbs by advancing through the cribriform plate; using the olfactory nerves for guidance[2] . Proliferation of the trophozoite begins in the olfactory bulbs, followed by host-cell interactions that trigger an inflammatory response and necrosis. Once inside the brain, N. fowleri proliferates and initiates cytotoxic effects that cause inflammation and necrotic hemorrhaging. The amoebastomes, pseudopodial food-cup projections, mediate the host-tissue invasion and ingestion[10] . NFa1 is thought to be the key protein that mediates the ability of N. fowleri for adherence, locomotion, and the formation of food-cups for ingestion. Shin et al showed that NFa1 was abundantly expressed and localized around the pseudopodial projections and food-cups[10] .

Entry through the nasal cavity is predominantly the main form of transmission; however, in some cases N. fowleri can enter through the ear when the tympanic membrane is ruptured. In addition, PAM may be contracted by dust containing N. fowleri. The infection is contracted when contaminated water is forcibly drawn into the nasal cavity.
[4,5] .

1.2 Clinical Manifestation and Symptoms

PAM is a progressive form of meningoencephalitis with a rapid onset. Symptoms typically become apparent 2-15 days after exposure. However, in some cases the onset of illness can be within 24 hours[2] .
. The clinical symptoms of PAM are similar to those seen in viral or bacterial meningitis. In fact, PAM is very difficult to diagnose because it does not have any distinct clinical features of its own [3]. Symptoms that occur early on in the disease include localized headaches in the frontal and temporal regions of the head, fever, vomiting, rhinitis, and a stiff neck[5] . The symptoms become more severe as the disease progresses. For instance, a patient will present with neurological abnormalities, a comprised mental state, diplopia, photophobia, seizures, and coma. The patient typically dies within one week after the onset of symptoms. Early diagnosis and treatment are critical for survival[2] .


1.3 Diagnosis

The current methods for diagnosing PAM are based on the identification of N. fowleri in clinical samples.

1.3a Histological and Morphological Assays

Wet-mounted Microscopy

Wet-mounted microscopy is used to examine the cerebrospinal fluid (CSF) for the presence of trophozoites.The technique involves phase-contrast microscopy and the ability to identify the morphological and locomotive characteristics of the trophozoite[11] . A typical trophozoite is about 10-25µmin size with a large nucleolus located in its center. The pattern of locomotion is usually rapid and directional, involving the use of pseudopodia[2] . A drawback to this technique is the physician's inability to identify the trophozoite, because it is often mistaken for a leukocyte[5] .

Flagellation Test (FT)

The flagellation test is a supplementary diagnostic tool used for detecting N. fowleri. The trophozoites are placed in a hypotonic solution and examined to see whether they convert to the flagellate state. The test is conducted a second time using a higher temperature to differentiate between different species of Naegleria. The drawback to this technique is an incidence for false negative results. Consequently, an additional tool is used regardless of the outcome. For instance, polymerase chain reaction (PCR), ELISA, and restriction length fragment polymorphism (RFLP)[12] .

Cell Culture

Cell culture examination is subsequently used if N. fowleri is suspected in wet-mount microscopy. The procedure for this test involves incubating a small drop of sedimented CSF after being mixed with 1mL of sterile water and placed in a tube. The incubation lasts for about 3 hours at 37 degrees Celsius. The physician must examine the culture regularly for the presence of actively mobile pear-shaped flagellates; typically composed of two terminal flagella. In addition, the CSF is typically grayish or yellowish-white during the early phase of infection. As the disease progresses the CSF turns red as the amount of red blood cells increases[4,5] .

1.3b Immunological Assays

Enzyme-linked Immunosorbent Assay (ELISA)

Several immunological assays have been developed to identify N. fowleri. These methods include immunoelectrophoresis and indirect immunofluorescence staining. Recently, the enzyme-linked immunosorbent assay(ELISA) was adopted for the detection of N. fowleri using a specific monoclonal antibody (Mab 5D12)[8] . ELISA is capable of distinguishing N. fowleri from other species of Naegleria and detecting it in all three stage of its lifecycle. The drawback is the requirement of a cell culture before ELISA can be performed[13] .

1.3c Biochemical and Molecular Assays

Polymerase Chain Reaction (PCR) Assay

Polymerase chain reaction protocol are currently the best diagnostic tools for PAM. The protocols are comprised of conventional, nested, multiplex, and real-time PCR methods. Recently, Obernauerova et al developed a novel addition to the real-time PCR diagnostic protocol that allows for fast and accurate detection of N. fowleri. The addition is a fluorescent labeling probe that selectively targets a gene unique to N. fowleri called Mp2Cl5.[14]

1.3d Issues with Diagnosing PAM

There are 3 main issues that make the diagnosis of PAM rather difficult. Patients that contract PAM do not have any distinct features that differentiate the infection from other forms of viral or bacterial meningitis. In addition, awareness among clinicians is low because the disease is very rare. Lastly, the disease is highly fatal with a rapid onset. As a result, the right diagnosis is often too late for effective treatment.

1.4 Pathophysiology

1.4a Inflammation and Purulent Exudate

Inflammation is one of three main features that underlie the pathophysiology of PAM. Inflammation initiates in the olfactory bulbs, and eventually spreads to other areas of the brain and meninges. According to one study, the inflammation is limited only to the frontal regions of the brain. The current understanding regarding the N. fowleri-induced inflammatory process initially involves two types of cell-cell interactions. The first involves N. fowleri-astroglial cell interactions and the activation of Interleukin-8 (IL-8) genes through the ERK-1/2 signaling pathway[15] . N. fowleri stimulates the activity of (1) extracellular signal-regulated kinases (ERKs) and the (2) DNA binding activity of activator protein-1 (AP-1) to upregulate the expression of IL-8 genes in human astroglial cells. The IL-8 genes produce cytokines that play a key role in the development of the inflammatory response against the trophozoite[16] . N. Fowler-microglial cell interactions are also implicated in the process of inflammation. Pro-inflammatory cytokines, such as interleukin-6, IL1-beta, and tumor necrosis factor-alpha, are released when N. fowleri interacts with microglia[3] . Purulent exudate is typically associated with the inflammatory process. The exudate accumulates within the base of the brain, olfactory bulbs, brainstem, cerebellum, and in between sulci, causing congestion and hyperemia

in the leptomeninges[2] .

1.4b Brain Hemorrhaging and Necrosis

The hemorrhagic and necrotic events are mediated by N. fowleri-host cell interactions. A series of studies examining the interaction between N. fowleri and mammalian cells have shown that various proteins are involved.The trophozoite induces necrotic hemorrhaging by releasing cytolytic proteins and by trogocytosis. These mechanisms are used to destroy neurons and other cell types for easy digestion. The mechanism selected by the trophozoite depends on its pathogenic strength, at least in vitro. For example, weakly pathogenic trophozoites use trogocytosis, a mechanism involving the ingestion of mammalian cells, such as neurons, with a 'food-cup' structure located on the surface of the trophozoite. In contrast, highly pathogenic trophozoites consume mammalian cells after releasing cytolytic proteins[8] .

N. fowleri secretes enzymes that degrade a wide variety of connective tissue and structural proteins. For instance, N. Fowleri degrades sphingomyelin by releasing proteases, acid hydrolases, phospholipases, and phospholipolytic enzymes. In addition, the trophozoite secretes neuraminidases and elastase. These enzymes are responsible for degrading collagen and proteoglycans, and for altering glycolipid and phospholipid composition to induce demyelination, respectively. Since the cytopathology of PAM is poorly understood, the proteins aforementioned are by no means an exhaustive list and the exact determinants of the pathogenicity remain unclear[8] .

Neuronal lysis by the trophozoite triggers an inflammatory response, which ultimately leads to the destruction of brain tissue. The inflammatory response is associated with necrotic hemorrhaging, resulting in widespread lesions through the cortex and spinal cord. The areas of the CNS most susceptible to the hemorrhaging include the base of the brain, olfactory bulbs, temporal and orbitofrontal lobes, hypothalamus, brainstem, and cervical portion of the spinal cord
[2] .

1.5 Treatment

1.5a Amphotericin B (AMB) Therapies

There is no cure for PAM at present (shin et al). The primary therapeutic agent for PAM typically involves a polyene antifungal drug called amphotericin B (AMB). It is administered alone or in conjugation with other drugs [3]. For instance, AMB has been administered with imidazole and rifampicin drugs, such as miconazole, fluconazole, and ornidazole[2,5] . Treatment is usually limited to 10 days because of adverse side effects, such as electrolyte imbalances, kidney damage, and hematopoietic changes that result in anemia[2] . Consequently, many agents, including antifungal, antiprotozoal, antibacterial, and antipsychotic drugs, have been screened for therapeutic activity against N. fowleri; in vivo and in vitro[16] .

1.5b Novel Research in Therapeutic Strategies

In 2002, Brenner et al conducted a series of studies to test the amoebicidal activity of a macrolide antibiotic, called azithromycin, against N. fowleri in a mouse model of PAM and in vitro. The study concluded that azithromycin was highly active against N. fowleri[17] . An additional study in 2007 investigated the effect of combining AMB and azithromycin in vitro and in mouse models of PAM. A synergistic effect was produced against N. fowleri when AMB and azithromycin were used in conjunction. Azithromycin is believed to suppress the synthesis of bacterial proteins and inhibit translocation by binding to the 50S ribosomal subunit[18] . However, the exact mechanism of action against N. fowleri is not clear.

A recent study conducted by Shin et al, also investigated the efficacy of miltefosine and chlorpromazine against N. fowleri in both in vivo and in vitro models. Chlorpromazine and miltefosine both demonstrated amoebicidal activity and repressed the proliferation of N. fowleri in vitro. Chlorpromazine had the highest clinical efficacy against N. fowleri infection in vivo. A comparative analysis showed that chlorpromazine was less toxic and more effective than AMB when treating for infection both in vivo and in vitro[19]

The Nfa1 gene, responsible for mediating the pathogenicity of N. fowleri, is another promising target for developing a novel therapeutic strategy. Cytochalasin D, an actin polymerization inhibitor, was shown to be effective in treating against N. fowleri by reducing its ability to form an amoebastome in vitro. In addition, an antisense oligonucleotide of the Nfa1 gene and an anti-Nfa1 polyclonal antibody produced the same effects[20] . Most recently, RNA interference (RNAi) was used to down-regulate the levels of Nfa1 mRNA and protein. This resulted in a lowered cytotoxic effect on macrophages in vitro[21] .

1.6 Epidemiology

In 1965, M. Fowler and R.F. Carter identified and described N. fowleri as a pathogenic species causing infection in humans[2,4] . Since then, only 235 cases of PAM have been reported worldwide in over 16 countries, making the disease rare[4] . In over 95% of cases, the individual has died due to the severe complications caused by inflammation and hemorrhagic necrosis. The disease affects young healthy individuals with recent exposure to water contaminated with N. fowleri. The estimated occurrence for PAM is unreliable, however, the incidence of PAM is highest in young males; possibly due to a higher rate of males engaging in recreational water-based activities.[2,16] .

1.6a Worldwide Occurrence

Cases in Africa
Only six cases of PAM were reported in Africa: four in Nigeria and one in both Namibia and Madagascar. The cases in Nigeria were particularly unique because it was hypothesized that three of four patients contracted the infection from dust containing cysts[4] .

Cases in Asia
In Asia there have been 39 confirmed cases of PAM: 17 patients in Pakistan, 12 in Thailand, 7 in Indian, two in Japan, and one in China. In Pakistan the disease was contracted from the use of unfiltered tap water during a 17-month period. In Thailand, India, and China the disease was contracted from swimming in lakes, canals, hot springs, or thermally polluted waters. In Japan, the source of infection is unknown[4] .

Cases in Australia
The 19 reported cases of PAM in Australia occurred in a town located in the Southern state. The town's water supply was contaminated with N. fowleri. In New Zealand, nine patients contracted the disease after swimming in geothermal water[2] .

Cases in Europe
There have been 24 confirmed cases of PAM reported in Europe: specifically in the Czech Republic, Belgium, Italy, and the UK. The disease was contracted either in an indoor swimming pool, geothermal bath, or in a stream thermally polluted by the effluents of an industrial plant. For instance, 16 patients in the Czech Republic and four in Belgium contracted the disease in an indoor swimming pool; however, investigators could not find traces of N. fowleri from the swimming pools involved in Belgium. In addition, one patient in Italy contracted the disease after swimming in a river and three others in the UK after swimming in a geothermal bath. While no other countries in Europe have reported cases of PAM, N. fowleri has been found repeatedly throughout the rest of Europe, mainly in France[4] .

Cases in North and South America
There have been 120 confirmed cases of PAM in North America: 111 cases have been reported in the USA, and nine in Mexico. In the USA, N. fowleri infection predominately occurs in the southern states. Most patients contracted the disease after swimming in warm freshwater bodies. In South America, PAM was reported only in Venezuela and Brazil, seven and five cases respectively. In Venezuela, 7 patients contracted the disease after swimming in natural warm lakes or untreated pools. In Brazil, five cases were linked to an artificial lake[1,4,5] .
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