Running head: PRIMARY AMOEBIC MENINGOENCEPHALITIS 1.

Primary Amoebic Meningoencephalitis: An Overview

Mario Rodriguez Gonzalez

Miami Dade College

Author note

Mario Rodriguez Gonzalez, Infectious Diseases and Control Practices: Class 6198,

Medical Campus, Miami Dade College.

Contact: [email protected]
PRIMARY AMOEBIC MENINGOENCEPHALITIS 2

Abstract

This paper is an overview of Primary Amoebic Meningoencephalitis (PAM) caused by a free

living amoeba species, Naegleria fowleri, usually referred in the media as ‘the brain-eating’

amoeba. Warm freshwater environments and soil are the preferred habitat for N. fowleri.

Individuals, usually children, who swim or otherwise get in contact with a N. fowleri infected

freshwater source, get infected when the amoeba enters the brain through the nasal passage. Due

to lack of specific diagnostic data, tests, and the low incidence of the disease around the globe,

which in itself lowers the level of clinical suspicion, the condition usually is misdiagnosed and/or

treated inadequately. The reported survival rate is very low with only few survivors worldwide;

consequently, many of the reported cases were diagnosed post-mortem by autopsy. Accordingly,

PAM is a waterborne disease with a more than 97% case-fatality rate, affecting mostly children

(Capewell, Harris, Yoder, Cope, Eddy, Roy,…Beach, 2014).

Keywords: Primary Amoebic Meningoencephalitis, Naegleria fowleri, brain-

eating amoeba
PRIMARY AMOEBIC MENINGOENCEPHALITIS 3

Primary Amoebic Meningoencephalitis

General Description

Free-living amoebae are protozoa present in all regions of the world except Antarctica.

Free-living amoeba of three species, Acanthamoeba, Balamuthia, and Naegleria, are the

pathogens responsible for amoebic encephalitis. There are two types of amoebic encephalitis:

Granulomatous Amebic Encephalitis (GAE) known to be caused by Acanthamoeba and

Balamuthia species, and Primary Amoebic Meningoencephalitis (PAM) which is described as an

acute, rapidly progressing, necrotizing, and hemorrhagic meningoencephalitis caused by

Naegleria fowleri, frequently called ‘the brain-eating’ amoeba.

Epidemiology

The first case of PAM was described in 1965 (Fowler & Carter, 1965). In Fowler &

Carter’s (1965) report, there is a reference to a 1961 case where a 9-year-old boy in Australia,

died due to meningitis that was uninterestingly typical except for the presence in the brain and

meninges of large numbers of amoebae which differed morphologically from the enteric amoebic

parasite Entamoeba histolytica. In regards to the 1961 case, “No pathogenic organisms were

cultured from the cerebrospinal fluid or meningeal exudate, but the clinical record and laboratory

investigations were considered to be characteristic in all other respects of acute bacterial

meningitis” (Fowler & Carter, 1965). The Fowler & Carter’s (1965) report was written after

three more patients from the same location died in similar circumstances in 1965. Fowler &
PRIMARY AMOEBIC MENINGOENCEPHALITIS 4

Carter (1965) made a brief account of the clinical and pathological manifestations of the 1965

cases, and they hypothesized to be in the presence of a previously undescribed disease.

Around the world, there have been a minimum of 440 reported cases of PAM (Parija &

Wallace, 2019). As it is stated in the Parija & Wallace’s (2019) article, the risk of infection is

now estimated to be 1 case per 2.6 million exposures to N. fowleri. Cabanes, Wallet, Pringuez &

Pernin (2001) mention that the United States, Australia, and the Czech Republic were the

countries where most of PAM cases have been reported. In general, patients were mostly healthy

children or young adults, but the infectious freshwater source varied depending on the country:

in the case of the Czech Republic, Belgium, or New Zealand, patients had been swimming in

heated pools or thermal waters; in North America, they acquired the disease after swimming in

ponds, lakes, or rivers; and in Australia, patients used water colonized by amoebae from

domestic sources.

In 2018, Nepal reported the first PAM case where a patient acquired the disease without

having any contact with an environmental source of water (Baral & Vaidya, 2018). The case was

reported as a PAM case of “an elderly immune-competent male without environmental exposure

to freshwater, mimicking as herpes encephalitis” (Baral & Vaidya, 2018, Abstract). The report of

Baral & Vaidya (2018) further describes the patient as a hypertensive, otherwise healthy, male

who was not on any kind of immunosuppressant therapy and without a history of swimming in

his lifetime or recent records of traveling out of his country.

Cabanes et al. (2001) in their attempt to assess the risk of PAM for swimmers potentially

exposed to N. fowleri highlighted the lack of available information from humans on which they

could base “a quantitative assessment of the risk of PAM associated with swimming or other
PRIMARY AMOEBIC MENINGOENCEPHALITIS 5

recreational activity carried out in water containing N. fowleri” (Abstract, para. 5). Until 2001

when Cabanes et al.’s (2001) article was published, the only estimate was based on data from a

1977 Florida study (Wellings, Amuso, Chang, & Lewis, 1977). Wellings et al. (1977) reviewed

the epidemiological data gathered on five of the seven fatal primary amoebic

meningoencephalitis cases reported from 1962 until the date the study was initiated in January

1975. According to Wellings et al. (1977), the data confirmed the presence of pathogenic

Naegleria amoebae in four freshwater lakes in Florida. The Florida study reported a risk of 7

cases per billion swimming episodes in water (Wellings et al., 1977) for which no precise N.

fowleri concentration has been measured (but up to 40 N. fowleria amoebae/liter) (Cabanes et al.,

2001). In their report, Cabanes et al. (2001) affirm that their quantitative risk assessment lead the

French health authorities to “set a maximum level of 100 N. fowleri amoebae per liter, not to be

exceeded in watercourses where human exposure is possible” (Conclusion, para. 10).

In United States, there have been 145 PAM infections from 1962 through 2018 (Centers

for Disease Control and Prevention (CDC), National Center for Emerging and Zoonotic

Infectious Diseases (NCEZID), & Division of Foodborne, Waterborne, and Environmental

Diseases (DFWED), 2019). The US infection rate amounts to 0 to 8 infections per year, with a

historic prevalence in southern states (Capewell et al. 2014); although, in 2010, a case of PAM

associated with local recreational water exposure was reported for the first time in the northern

state of Minnesota (Kemble, Lynfield, DeVries, Drehner, Pomputius, Beach,…Danila, 2012).

Capewell et al.’s (2014) article indicates that PAM patient demographics showed a high

proportion of males (76%) and children (83% aged ≤18 years), with a median age of 12 years

(range, 8 months to 66 years). In 2019, the statistics of reported PAM cases by States, kept by the

CDC (2019), clearly confirms that exposure locations were predominantly situated in the south
PRIMARY AMOEBIC MENINGOENCEPHALITIS 6

and west regions of the United States, with Florida and Texas as the states with the highest

incidence of the disease nationwide. Yet again, Capewell et al. (2014) insists that beginning in

2010 and thereafter, the epidemiology of the disease changed with reports of northernmost PAM

cases. Capewell et al. (2014) mention that since the first 2010 case of PAM in the northern state

of Minnesota, there have been more cases in that same state and in other northern states like

Kansas and Indiana. As it is suggested by Capewell et al.’s (2014) article, the epidemiology of

the disease needs to reflect this shift in order to expand the network of clinicians that might

encounter PAM cases and, subsequently, heighten the level of alertness in those regions,

improving the rate of early case detection by considering PAM as a possibility in the differential

diagnosis of future meningoencephalitis cases.

Etiologic Agent

There are 30 identified species of Naegleria but only N. fowleri is known to be

pathogenic to humans. Although the genus has two other species, N. australiensis and N. italica,

identified as etiologic agents of infections in intranasally or intracerebrally challenged mice, they

have never been isolated from clinical specimens (Visvesvara, Moura & Schuster, 2007).

Naegleria fowleri are eukaryotic protists with an extensive global distribution, and

classified as amphizoic because they can survived as free-living microbes in nature and

occasionally be parasitic within host tissue (Visvesvara et al., 2007).

Morphology

Naegleria fowleri is an amoeba-flagellate, with three distinctive morphologies in its life

cycle: trophozoite (the infectious form), transitory, pear-shaped flagellate; and cyst (Capewell et

al., 2014; Visvesvara et al., 2007).

The trophozoite, size 10 to 25 µm, has a cytoplasm that shows a clear division between

the ectoplasm and the endoplasm; the latter encloses ribosomes, food vacuoles, several

mitochondria, and a contractile vacuole and trailing protoplasmic filaments delineating the uroid

or posterior end (Visvesvara et al., 2007).

The size of the temporary flagellate stage ranges from 10 to 16 µm. This stage is easily

recognized by its pear shape and the two flagella at the uroid (Visvesvara et al., 2007).

The spherical, double-walled cyst, with a 7-15 µm diameter, usually has a thick endocyst

surrounded by a thin ectocyst with pores in the cyst wall which are not always visible with the

light microscope because they are flush to the wall (CDC et al., 2017; Visvesvara et al., 2007).

Visvesvara et al. (2007) indicate that a visible single vesicular nucleus with a protruding,

centrally placed nucleolus is a common feature shared by the three stages.

Mobility

The trophozoite moves rapidly in an active sinusoid/limacine locomotion through

lobopodia, a pseudopodium with a core of endoplasm produced at the anterior end (Visvesvara et

al., 2007). In the flagellated stage, the microbe uses its two flagella
PRIMARY AMOEBIC MENINGOENCEPHALITIS 8

Life cycle

Trophozoites reproduce by binary fission during which their nucleus undergoes

promitosis, a type of mitosis where the nuclear membrane remains intact through the whole

process (CDC et al., 2017). When trophozoites encounter an adverse environment they can turn

into the temporary flagellated stage (CDC et al., 2017). If conditions improve, within an hour or

less, the pear-shaped flagellate, which doesn’t divide or feed, usually reverts back to the

trophozoite stage (CDC et al., 2017). The trophozoite transforms into a cyst when it confronts a

harsh environment with insufficient food sources and poor growth conditions (CDC et al., 2017).

The cysts are highly resistant to environmental pressures, like desiccation, in order to ensure the

microbe survival until there is a return to more favorable living conditions (CDC et al., 2017).

Ecology

Naegleria fowleri is a thermophilic organism that can live in environments with

temperatures up to 45°C. The optimal temperature of the water is said to be in a range between

25 to 44°C (Cabanes et al., 2001). For that reason, it is mostly during the warmer seasons of the

year when they proliferate (Visvesvara et al., 2007). Around the globe, Visvesvara et al. (2007)

affirm that amoebae have been recovered “from fresh-water pools, puddles, lakes, rivers,

swimming pools, hot springs, thermally polluted effluents of power plants, hydrotherapy pools,

aquaria, sewage, irrigation canals, and even from the nasal passages and throats of healthy

individuals” (Cultivation section, para. 1). Due to the microorganism sensitivity to high levels of

osmolarity, the only habitat that has never been colonized by the amoebae is seawater

(Visvesvara et al., 2007).

In clinical cases, Naegleria fowleri trophozoites have been isolated from the

cerebrospinal fluid (CSF) and tissue while flagellated forms are only sporadically present in the

CSF. There has never been confirmation of cysts present in brain tissue or fluids (CDC et al.,

2017).

Nutrition

In nature the amoebae feed upon bacteria. In clinical cases, the microbes attack the

central nervous system tissue.

Pathogenesis

Visvesvara et al. (2007) indicate that the amoeba enters the nasal passages of

unsuspecting victims while they are swimming or involved in other water contact activities. The

nasal mucosa invasion of the microbe is achieved by the secretion of a protein with mucinolytic

activity that degrades the nasal protective layer of mucus (Parija & Wallace, 2019). From the

nasal passages, Visvesvara et al. (2007) say, the microbe gains access to the central nervous

system (CNS) through the olfactory neuroepithelium, where it is believed the amoebae is

phagocytosed by the sustentacular cells lining it. According to Visvesvara et al. (2007), the

trophozoites arrive to the subarachnoid space through the cribriform plate in their way to the

microbe’s final destination, the brain parenchyma. Parija and Wallace (2019) identify the

foramen of Luschka and Magendie as the points of entry of trophozoites into the ventricular

system: they gain access to the choroid plexus where they destroy the ependymal layer of the

third, fourth, and lateral ventricles and produce acute ependymitis. The amoeba uses the pool of

glucose and protein in the CSF to grow and reproduce; the richly oxygenated medium in the CSF
PRIMARY AMOEBIC MENINGOENCEPHALITIS 10

and in the brain also contributes to the proliferation of the microorganism (Parija and Wallace,

2019).

The cells of the brain tissue undergo lysis due to several factors, as described by

Visvesvara et al. (2007): first, the amoeba feeds upon the neural tissue by producing sucker-like

appendages or amoebostomes; second, it is possible that the microbe destroys the cell

membranes by way of enzymes like phospholipase A and B which degrades cell membrane

phospholipids, neuraminidase or elastase that destroys the sugars and proteins part of the cell

membrane scaffolding; perforin-like, pore-forming proteins that punctures the neural cells; and,

finally, amoebic cytopathic proteins that elicit the death of susceptible tissue cells by promoting

the activation of their apoptosis pathway.

All the events previously described above results in a pus-filled meningitis and

encephalitis. Visvesvara et al. (2007) describe the severe lesions, and inflammation and

hemorrhagic events caused by the amebic proliferation throughout the CNS: edematous and

severely congested cerebral hemispheres, significantly congested, diffusely hyperemic and

opaque leptomeninges (arachnoid and pia mater); limited purulent exudate within sulci, the base

of the brain, brainstem and cerebellum; hemorrhagic necrosis of olfactory bulbs usually

surrounded by purulent exudate, numerous superficial hemorrhagic areas in the cortex, lesions

found in and around the base of the orbitofrontal and temporal lobes, base of the brain,

hypothalamus, midbrain, pons, medulla oblongata, and the upper portion of the spinal cord; and

obliteration of the cisternae around the midbrain and the subarachnoid space over the cerebral

hemispheres.
PRIMARY AMOEBIC MENINGOENCEPHALITIS 11

According to Visvesvara et al. (2007), the cerebral hemispheres, brain stem, cerebellum,

and upper portion of the spinal cord exhibit, microscopically, fibrino-purulent leptomeningeal

exudate containing predominantly polymorphonuclear neutrophils (PMN), a few eosinophils and

macrophages, and some lymphocytes; edematous and necrotic neural tissue with numerous

amoebic trophozoites usually in pockets without PMN’s around, and Virchow–Robin spaces

with trophic amoebae deep into those structures usually around blood vessels with no

inflammatory response. As it was said earlier, amoebic cysts have never been found in neural

fluids or tissue.

Clinical Manifestations

PAM’s incubation period is case dependent since it correlates to the microbial load and

the specific strain virulence (Visvesvara et al., 2007). Nevertheless, Visvesvara et al. (2007)

estimate that the time from initial water contact to the onset of the illness is usually 5 to 7 days,

and could even be just after 24 hours. Due to lack of specific clinical manifestations that could be

used to differentially diagnose PAM from acute pyogenic or bacterial meningoencephalitis,

clinicians must inquire about any contact with fresh water in the patient’s recent past (Visvesvara

et al., 2007).

The disease could be divided in two stages. The first is characterized by a sudden onset of

bifrontal or bitemporal headaches, high fever, nuchal rigidity usually accompanied with positive

Kernig and Brudzinski signs, followed by nausea, vomiting, irritability and restlessness

(Visvesvara et al., 2007). The second stage may be preceded by photophobia, followed by a

series of neurological clinical manifestations that signal an increase in brain damage, leading

eventually to death within a week; those symptoms include lethargy, seizures, confusion, coma,
PRIMARY AMOEBIC MENINGOENCEPHALITIS 12

double vision or bizarre behavior; and third, fourth, and sixth cranial nerves palsies, indicative of

brain edema and herniation (Visvesvara et al., 2007). The intracranial pressure usually climbs to

600 mmHg or higher and, in some cases, cardiac rhythm irregularities and necrosis of the

myocardium may occur (Visvesvara et al., 2007).

Management and Prognosis

The differential diagnosis of PAM must include acute bacterial meningitis, HSV-1

encephalitis, viral or fungal meningitis. In the case of immunosuppressed patients, clinicians

must also consider toxoplasmosis, cytomegalovirus infection or other opportunistic pathogens.

Detection

Although it is essential to count on specialized experience, immune-histochemical

staining, or polymerase chain reaction (PCR) tests in order to reach a definitive diagnosis of

PAM, a microscopic examination of the cerebrospinal fluid (CSF) immediately after collection

would allow detecting on a wet mount of the CSF actively motile trophozoites, which can be

further identified by staining CSF smears with Giemsa-Wright stains (Capewell et al., 2014).

Also, a bacterial culture test of the amoeba can be made to confirm the diagnosis by observing

the microbe feeding on the bacteria under a phase contrast microscope. Polymerase chain

reaction or next generation sequencing are fundamental tools in our hands to detect Naegleria

fowleri nucleic acids in the CSF and tissue specimens. Immunohistochemistry and indirect

immunofluorescence are also used to identify Naegleria fowleri antigens by directly staining the

amebic antigens present in the CSF and tissue specimens. Serologic or blood tests are also

performed to check proteins and leukocytes, which may be unusual, and to detect presence of

antigens.
PRIMARY AMOEBIC MENINGOENCEPHALITIS 13

Therapeutic Management of PAM

Capewell et al. (2014) reports that based on the available treatment data for 70 of the 142

(49%) patients in the United States, 36 (51%) of them were treated for PAM with only 3 (8%)

surviving the infection. Amphotericin B was the drug of choice in all 36 patients treated for

PAM; the way they received it varied: (7 [19%]), only intravenous (IV) therapy; 5 (14%), only

intrathecal (IT) therapy; and 24 (67%), a combination of IV and IT therapy (Capewell et al.,

2014). 7 patients were identified by Capewell et al. (2014) as treated with a non-deoxycholate

amphotericin B formulation that includes liposomal and lipid complex. In conjunction with

amphotericin B treatment, IV or IT azole therapy was also used in 21 (58%) patients, while oral

(PO) or IV rifampin was chosen for other 23 (64%) patients. The therapeutic data gathered in

89% (32 of 36) of the PAM treated patients showed 3 days (range, 0.75–7 days) as the median

time from onset to the start of PAM therapy, and 0.75 days (range, 0–4 days) as the median time

from hospitalization to the start of PAM therapy (Capewell et al., 2014). During the hospital

stay, Capewell et al. (2014) affirms that 94% of the patients were treated with antibiotics against

bacterial meningitis pathogens. Several therapeutic agents were tried in the patients that didn’t

receive any treatment for PAM (Capewell et al., 2014).

In 2013, the US had its first 2 PAM survivors in 35 years, so the recent therapeutic

management of PAM has evolved in accordance (Capewell et al., 2014). Out of the 32 fatalities

with available clinical data, 25 (78%) were treated for PAM 3 days from the onset of symptoms;

therefore, Capewell et al., 2014 suggest that the survival rate among PAM patients depend

probably on several elements besides treatment and time of diagnosis, for example, microbial

load and virulence. The 2013 survivors were given all the drugs that a 1978 survivor received,

including fluconazole, used in place of miconazole, and adding miltefosine to the original
PRIMARY AMOEBIC MENINGOENCEPHALITIS 14

treatment, a drug on clinical trials back then, along with azithromycin (Capewell et al., 2014). In

addition to the drug therapy, the elevated intracranial pressure was also aggressively managed

with external ventricular drain placement, therapeutic hypothermia, and administration of

steroids (Capewell et al., 2014).

Prognosis

As we stated earlier in this paper, PAM is a deadly disease with a high fatality rate of

almost 97%; for that reason, in order to increase the survival rate of PAM patients we are

compelled to continuously seek and trial new effective therapies against N. fowleri (Capewell et

al., 2014).

Treatment and Prevention

Treatment

Clinical experience and lab experiments indicate that traditional therapy of IV/IT

amphotericin B, IV/IT azole drugs, with or without PO rifampin, is to be recommended: the

treatment was used in the survival cases of the US and Mexico patients and, in vitro, the drugs

have shown anti-amoeba activities against N. fowleri (Capewell et al., 2014).

In spite of the continued use of amphotericin B as the drug of choice, its limited efficacy

is evidenced by the fact that only 3 (8%) of 36 patients treated with this drug survived (Capewell

et al., 2014). Amphotericin B and miconazole were found to have an additive effect in studies of

in vitro drug-sensitivity of amoebas isolated from the original 1978 survival case (Capewell et

al., 2014). As reported by Capewell et al. (2014), miconazole was shown to be inhibitory of

Naegleria proliferation in a separate in vitro study. Visvesvara et al. (2007) report the specific
PRIMARY AMOEBIC MENINGOENCEPHALITIS 15

case of a Californian girl that survived the infection thanks to an aggressive therapy consisting

on IV and IT amphotericin B, IV and IT miconazole, and oral rifampin. After 4-year of the

infection, the patient shows no signs of neurological abnormalities, and she is free of any other

related or unrelated health condition (Visvesvara et al., 2007). There was apparently a synergistic

effect between amphotericin B and miconazole although it is generally considered that rifampin

had no therapeutic usefulness against the microbe (Visvesvara et al., 2007).

The 2013 survivors were treated with miltefosine, a drug whose in vitro efficacy against

Naegleria has been proved and has been used somewhat effectively to treat infection caused by

the related amoebas Acanthamoeba and Balamuthia (Capewell et al., 2014). CDC recommends

the drug as a first line treatment for free-living amoebae infections including primary amoebic

meningoencephalitis (CDC, 2019).

Capewell et al. (2014) propose to add azithromycin as part of the PAM treatment regime

because it was also used in the 2013 US survival cases, and its proven efficacy against N. fowleri

infection in both a mouse model and in vitro. It should also be noted that the synergy between

azithromycin and amphotericin B can increase the effectiveness of any PAM treatment by way of

their combined effects (Capewell et al., 2014).

Prevention

As per CDC (2019), there is no precise estimate of the true risk of PAM due to the lack of

significant data: the annual rate of the infection in the US is 0-8 cases contraposed to hundreds of
PRIMARY AMOEBIC MENINGOENCEPHALITIS 16

millions of people, in the same time span, swimming in environmental warm freshwater in the

country. The question about why of the millions of people exposed to warm recreational fresh

waters, only but a few become infected remains unanswered (CDC, 2019). Although there have

been several studies trying to estimate the minimum environmental concentration of Naegleria

fowleri that can pose a risk to humans, the CDC (2019) affirms that, at present, we do not have a

method to measure in a reproducible and accurate way the amoeba water concentration, so it

remains to be seen how to come up with a standard to protect people from the disease, and how

public health authorities could stablish a surveillance system based on that standard and follow

necessary health protocols to enforce it.

PAM is a defined as a rare disease because of the very low risk of acquiring a Naegleria

fowleri infection: only 34 reported infections from 2009 to 2018 in the US; in contrast, from

2001 to 2010, the US had more than 34,000 drowning deaths. Nevertheless, there must be an

increase in the alertness of all healthcare providers throughout the country due to PAM

incidences in locations historically excluded from the classical disease epidemiology, e.g.

northern states (Capewell et al., 2014). Also, traveling is one of the issues that need special

attention in PAM diagnosis because the disease onset and management occurs in one place, but

the exposure happened to be in another location patients just recently visited (Capewell et al.,

2014). Therefore, Capewell et al. (2014) suggest that even in cases of patients with flu-like or

bacterial meningitis symptoms, clinicians should make it a habit to inquire about any recent

event in patients’ record that might indicate they were exposed to amoebic contaminated warm

freshwater.

Prevention must be centered in education by providing people using recreational or any

other source of warm freshwater with accurate information about the disease, so they can
PRIMARY AMOEBIC MENINGOENCEPHALITIS 17

recognize any sign of possible amoebic infection after their exposure to such a source. The

information should focus on the known sources of amoebic infections like lakes, rivers, thermal

waters, etc; and the microbe’s route of infection, the nasal passage. Humans cannot get infected

from oral intake of water contaminated with Naegleria and, although Naegleria fowleri “can

grow in pipes, hot water heaters, and water systems, including treated public drinking water

systems,…” infections occur “…very rarely when people submerge their heads, cleanse their

noses during religious practices, or irrigate their sinuses (nose) using contaminated tap or faucet

water” (CDC, 2019, Prevention & Control, para. 1).

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PRIMARY AMOEBIC MENINGOENCEPHALITIS 18

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PRIMARY AMOEBIC MENINGOENCEPHALITIS 19

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