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Received: 12 June 2019    Revised: 7 October 2019    Accepted: 12 October 2019

DOI: 10.1111/1440-1681.13192

REVIEW ARTICLE

Naegleria fowleri: Sources of infection, pathophysiology,

diagnosis, and management; a review

Muhammad Jahangeer1 | Zahed Mahmood1 | Naveed Munir1,2 |

Umm‐e‐Amara Waraich3 | Imtiaz Mahmood Tahir2  | Muhammad Akram4 |
Syed Muhammad Ali Shah4  | Ayesha Zulfqar1 | Rida Zainab4

1
Department of Biochemistry, Government
College University Faisalabad, Faisalabad,
Pakistan
2
College of Allied Health
Professionals, Directorate of Medical
Sciences, Government College University
Faisalabad, Faisalabad, Pakistan
3
Department of Biochemistry, University of
Agriculture, Faisalabad, Pakistan
4
Department of Eastern
Medicine, Directorate of Medical
Sciences, Government College University
Faisalabad, Faisalabad, Pakistan
Correspondence
Naveed Munir, Department of Biochemistry,
Government College University Faisalabad,
Faisalabad, Pakistan.
Email: naveedmunir.pk@gmail.com
Abstract
Naegleria fowleri, a thermophilic flagellate amoeba known as a “brain‐eating” amoeba,
is the aetiological agent of a perilous and devastating waterborne disease known
as primary amoebic meningoencephalitis (PAM), both in humans as well as in ani-
mals. PAM is a rare but fatal disease affecting young adults all around the world,
particularly in the developed world but recently reported from developing countries,
with 95%–99% mortality rate. Swimmers and divers are at high risk of PAM as the
warm water is the most propitious environment adapted by N. fowleri to cause this
infection. Infective amoeba in the trophozoite phase enter the victim's body through
the nose, crossing the cribriform plate to reach the human brain and cause severe
destruction of the central nervous system (CNS). The brain damage leads to brain
haemorrhage and death occurs within 3–7 days in undiagnosed cases and maltreated
cases. Though the exact pathogenesis of N. fowleri is still not known, it has exhibited
two primary mechanisms, contact‐independent (brain damage through different pro-
teins) and contact‐dependent (brain damage through surface structures food cups),
that predominantly contribute to the pathogen invading the host CNS. For the man-
agement of this life‐threatening infection different treatment regimens have been
applied but still the survival rate is only 5% which is ascribed to its misdiagnosis, as
the PAM symptoms closely resembled bacterial meningitis. The main objectives of
this review article are to compile data to explore the sources and routes of N. fowleri
infection, its association in causing PAM along with its pathophysiology; latest tech-
niques used for accurate diagnosis, management options along with challenges for
Pakistan to control this drastic disorder.

KEYWORDS
brain haemorrhage, diagnosis, management, Naegleria fowleri, primary amoebic
meningoencephalitis (PAM), vaccine, water borne disease

The peer review history for this article is available at https​://publo​ns.com/publo​n/10.1111/1440-1681.13192​

Clin Exp Pharmacol Physiol. 2020;47:199–212. © 2019 John Wiley & Sons Australia, Ltd |  199
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200       JAHANGEER et al.
1 |  I NTRO D U C TI O N
Naegleria fowleri is a free‐living, thermophilic, pathogenic flagellate
amoeba belonging to the Heterolobosea class. In warmer months of
the year, N. fowleri proliferates as it has the ability to tolerate tem-
peratures up to 45°C and feeds predominantly on bacteria on living
in natural bodies of warm freshwater, from where it has been fre-
quently detected.1,2 N. fowleri is also known as amphibolic amoeba
and there are three morphological stages of Naegleria species life
cycle have been identified: trophozoite (10–25  mm), pear‐shaped
temporary flagellate stage (10–16 mm) and cyst stage (8–20 mm).3,4
Depending on the environmental conditions, N. fowleri can
change its phenotype, and trophozoite is the reproductively‐active
stage which it exhibits under favourable conditions and it is consid-
ered as the infective stage. It has a single nucleus and multiplies by
binary fission; the nuclear membrane remains intact during this divi-
5
sion (a process known as pro mitosis). The appropriate temperature
for the best growth of this stage is 35–46°C. Trophozoites transform
to a temporary motile stage called flagellate when nutritional de-
ficiency occurs in the environment, but water is present, and this
stage thrives best in the temperatures rang 27–37°C. Under unfa-
vorable or adverse conditions, the metabolically dormant cyst stage
originates from the trophozoites and can survive in very low tem-
peratures. Both flagellate and cyst forms of N. fowleri are non‐re-
productive and non‐feeding stages, while only the trophozoites can
6,7
reproduce, feed, and/or become cysts.
Being a free‐living protist, N. fowleri feeds mainly on bacteria,
both Gram‐positive and Gram‐negative, as well as on algae, and
yeast. The response shown by N. fowleri to bacteria is through the
formation of food cups, chemotaxis and chemokines. 6,8
N. fowleri
is the only species that causes a lethal brain infection called pri-
mary amoebic meningoencephalitis (PAM), while over 40 species
of Naegleria have been identified. Though PAM is rare, it is a fatal
9,10
human disease with a mortality rate of 95%–97%. The death
occurs within approximately seven days, as it is a lethal, necrotiz-
ing, fulminant, and haemorrhagic meningoencephalitis.11 PAM oc-
curs significantly in immunologically strong individuals as well as in
healthy children and young adults, having recent exposure to recre-
ational freshwater.12 PAM is a waterborne disease, so most cases are
associated with diving and swimming activities in less chlorinated
pools, polluted canals and spas or during leisure sports such as water
skiing in contaminated environmental water sources, and the use of
3
neti pots for nasal cleansing and ablution.
This acute infection is developed by the entry of N. fowleri
through nasal cavity when water is forced or splashed into the nose.
N. fowleri causes destruction of neurons and explains why this is
also known as the “brain‐eating amoeba”; this term shows that the
enzymes and toxins of this parasite are typically involved in the de-
struction (eating‐up) of the brain.13,14 PAM is characterized by similar
signs and symptoms to those of viral or bacterial meningitis including
fever, headache, stiff neck, vomiting, anorexia, seizures, ultimately
death typically occurs within 3–7 days after the appearance of these
signs and symptoms.15 The virulence of the strain and the size of the
inoculums are involved in determining the time span between initial
contact with the pathogenic N. fowleri and the appearance of clinical
signs and symptoms that may vary from 2 to 3 days to up to as long
as 7–15 days.16
PAM is a deadly and rapidly spreading disease and many of the
cases have been reported in the USA, as described by Anser and
Hasan,17 where most of the people were involved in a swimming ac-
tivity. Recently PAM cases in Pakistan have been reported mostly
in males, but not in females, because of ritual rinsing of the nose
for ablution commonly with contaminated water, although in reli-
gious practice this action decreased chances of various infections.
In Pakistan the incidence of PAM in males and females may vary
and has been reported as a 2:1 ratio, which is different from that in
the US, where the predominance of PAM in females (63% of cases)
has been observed, while the median age of this acute infection
is 11  years (range from 4 to 56  years).5 Most PAM cases were re-
ported during summer in the United States, though in the winter
months, in northern states as well as in southern states with colder
waters such as Connecticut and Pennsylvania, N. fowleri presence
has been recorded but not even a single case has been found be-
tween December and March. Further, more recently, PAM cases
were reported from Karachi, Pakistan.18 This review article has been
compiled to summarize the possible risk factors for this infection,
pathogenesis, diagnosis and management strategies. Further, the
burden and challenges for Pakistan to handle this disease are also
described briefly.
2 | H I S TO R I C A L BAC KG RO U N D A N D
E PI D E M I O LO G Y O F PR I M A RY A M O E B I C
M E N I N G O E N C E PH A LITI S
The first case of PAM was reported in Florida, USA, in 1962. The
name Naegleria fowleri was given to it after the discovery of the
disease primary amoebic meningoencephalitis (PAM) in Australia
in 1965 by Malcom Fowler of the Adelaide Children's Hospital,
Australia, and R. F. Carter, who first described this disease. The
name “primary amoebic meningoencephalitis” was given to it in
1966 by Butt and then in 1968 by Carter to differentiate it from
the rare brain invasion caused by Entamoeba histolytica. PAM cases
have been reported from 15 countries all over the globe, excluding
Antarctica,12,19 numbering only a few hundred and are mostly from
Europe, Australia, and the United States, as well as from some Asian
countries.6 Though the number of annually reported infections re-
mained stable (0–8), recent changes in the epidemiology of PAM are
of great concern. A PAM case from the northern state of Minnesota
was reported for the first time in 2010, then in 2011 and 2012 fol-
lowed by additional cases from Indiana, Minnesota, and Kansas; this
led to increased concerns about an expanding geographic range of
this infection caused by the heat‐loving, potentially climate‐sensitive
pathogen. 20
The number of PAM patients reported from 1937 to 2013 was
142, in the United States. A vast study has revealed that from 1962
JAHANGEER et al.
to 2014, throughout the world approximately 260 cases have been
observed which include: 132 cases from the United States, 19 in
Australia, 17 cases from Pakistan, 16 cases from the Czech Republic,
11 from India, 9 in Mexico, 9 from New Zealand, 7 from Venezuela,
5 cases each from Thailand and Belgium, 4 from Nigeria, 2 from the
United Kingdom, and 1 case only from each of Namibia, Iran, Costa
Rica, New Guinea, South Africa and Madagascar. It was also reported
that only 11 patients survived out of these 260 reported cases.17,21
According to recent studies 16 cases of PAM have been reported
in India, only four patients survived out of these, while three patients
had no water contact, so it becomes doubtful that the infection was
due to N. fowleri. The majority of other patients in India used ground-
water, puddles, or ponds for bathing or swimming. N. fowleri pres-
ence has been confirmed in pond water, swimming pools and sewage
canals of India.7,22 It was also reported in other countries like China,
Hong Kong, Zhejiang, Zambia (Figure 1). 23,24
According to the US Center for Disease Control and Prevention
(CDC), the number of PAM cases in the United States from 1962
to 2015 was 138. Recently, in Asian countries, an increase in the
23
number of PAM cases has been investigated. With respect to the
occurrence of PAM, in the USA 143 cases have been reported from
1962 to 2016, 139 of which were fatal. 25 Around 440 cases are re-
corded worldwide. 26,27
3 | H A B ITAT A N D D I AG N OS I S O F
N .  fow l e r i
Naegleria fowleri, being a thermophilic protist, preferentially in-
habits warm water including lakes in the tropics and hot springs in
temperate zones. 28 However, N. fowleri trophozoites or cysts have
been classified depending upon its habitat in the most varied en-
vironments into two categories, natural habitats, and urban zones.
Natural habitats include hot springs and warm aquatic conditions,
F I G U R E 1   Worldwide reported cases of PAM due to Naegleria
fowleri until 2017
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ponds, freshwater lakes, and rivers while urban zones include con-
taminated and uncontaminated water sources, drinking water dis-
tribution system (DWDS) through pipe wall biofilms, 29 hotel and
domestic swimming pools, recreational fountains, hospitals, geo-
thermal heated water, contaminated drinking water and water parks,
dental unit waterlines (DUWLs), and nasal flooding using tap water.
The number of N. fowleri is greater in sites having increased water
temperature that is above 28ºC.30 Hence, environmental monitor-
ing by using accurate and fast screening is essentially required as
risk prevention for discrimination between other free‐living amoeba
(FLA) and N. fowleri in water samples.31,32
The crucial points while fighting N. fowleri includes its detec-
tion and dispersal mode as both non‐pathogenic and pathogenic
species present in the environment. There are different methods to
specifically identify N. fowleri such as immunological or molecular,
other recent methods are also included visually detecting N. fowl-
eri. 27,33 Molecular based techniques like polymerase chain reaction
(PCR) are more sensitive, rapid and specific as compared to culture
and microscopic techniques.32 The direct quantification and de-
tection of microorganisms in environmental samples can be done
particularly by quantitative PCR (qPCR), without requiring cultural
isolation and such real‐time PCR‐based diagnostic methods might
become efficient and useful tools when implemented into routine
laboratory practice. 34-36 The pathogenicity test in mice and growth
temperature testing at a maximum of 45°C were used at first for
the identification of N. fowleri isolates. Later for the identification
of N. fowleri, isoenzymes and antibodies were used. The typing of
different strains of N. fowleri and Naegleria spp. was done by using
electrophoretic karyotyping (KE) and restriction‐fragment‐length
polymorphism (RFLP). Experimental work shows that from Europe,
Australia, USA, and New Zealand, the N. fowleri strains differ in iso-
enzymes, KE as well as in RFLP.8
The one nucleotide base pair difference in the 5.8 S rDNA and
the difference in internal transcribed spacer 1 (ITS1) length were the
basis for the detection of eight different types of N. fowleri.8,37 The
intra‐species variability was firstly observed by RFLP, it can also be
investigated by using molecular markers and then a higher level of
diversity is displayed by random amplification of polymorphic DNA
(RAPD). Therefore, five main variants, namely South Pacific (SP),
Cattenom (CAT), Chooz (CHO), Widespread (WP) and Euro‐American
(EA) which are not always correlated with geographical origin, have
been observed by using RAPD markers and microsatellite markers. 27
The rapidly evolving and a practical application of mass spectrometry
(MS) is the matrix‐assisted laser‐desorption‐ionization–time‐of‐flight
mass spectrometry (MALDI–TOF MS) that is used for strain differ-
entiation and rapid identification of microorganisms.38 However, the
use of MALDI–TOF MS for characterization of this organism is re-
ported in only a few reports. 2 One study has successfully applied
MALDI–TOF MS for the differentiation of Naegleria species and iso-
lates them along with statistical analysis. However, more robust bio-
marker identification is yielded by the combined MS visual analysis,
proteomics and fingerprinting approach, by using a database search
that allowed isolate differentiation among N. fowleri isolates.38
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202       JAHANGEER et al.

F I G U R E 2   Diagrammatic representation of N. fowleri pathophysiology. (1) From contaminated water, N. fowleri entered the nose. (2) N.
fowleri trophozoites move upward to enter the brain, (3 & 4) The olfactory mucosa and olfactory bulb are the first targets for N. fowleri.
(5A) Bluish shade indicates the expression of GPCR NF0059410 on Trophozoites of N. fowleri which is structurally homolog to the human
muscarinic M1 receptor (5B) model for GPCR NF0059410 a receptor for acetylcholine binding, (5C) sequence of amino acids of the N.
fowleri protein NF0059410 for the binding of acetylcholine. (6) Binding of acetylcholine to GPCR NF0059410 instead of the human receptor
generates neurotropic chemotaxis in the trophozoites which cross and damage the cribriform plate on moving upward and this movement
is further enhanced by the secretion of acetylcholine (red lines represent the action and secretion of acetylcholine from brain & olfactory
nerves

4 |  PATH O PH YS I O LO G Y O F N .  fow l e r i
I N FEC TI O N
The acute infection in the human body is developed when water
containing N. fowleri is forcefully inhaled into the upper nasal pas-
sages during bathing, swimming, or other recreational activities.18
This infection occurs firstly by attachment of amoeba to the nasal
mucosa, which then moves along the olfactory nerve and reaches
the olfactory bulbs through the cribriform plate within the CNS
(Figure 2). Furthermore, there is a possibility that the risk of PAM
is higher among those children and young adults that have more
porous cribriform plates.11,39 Research has revealed that infection
cannot be initiated by drinking contaminated water.40 Another study
indicated that this amoeba can only infect a person through the ol-
factory route, not by the oral route. There are certain events that
may be significant in disease progression such as the trophozoites
ability to attach to the nasal mucosa, a chemotactic response to
components of nerve cell, an increased locomotion rate.41,42
Clinical symptoms such as a change in the smell sense and re-
spiratory infection signify involvement of olfactory epithelium and
neural tissue invasion. 2 The signs and symptoms shown by patients
affected by PAM do not include any symptom of nasal inflammation
such as nasal pain, bleeding, tenderness at the nose bridge, sneezing,
and/or persistent rhinorrhea, before developing signs of PAM such
as meningitis. Moreover, damage and destruction of the olfactory
bulb, olfactory mucosa and adjacent areas of the brain are mostly
observed after postmortem findings, while similar destruction in the
region of non‐olfactory mucosa of the nasal cavity is not observed.43
One study indicated that N. fowleri favours the frontal lobe as com-
pared to the parietal lobe. As N. fowleri adopted the nasal route for
JAHANGEER et al.
entry, showing that N. fowleri probably has a closed association with
frontal lobe, this is because the olfactory bulb has anatomical prox-
imity to the frontal lobe; in the nasal passage it is terminal to the
olfactory neuro‐epithelium, and hence traverses the cribriform plate
44,45
to the brain described diagrammatically in Figure 2.
The absence of any damage in the nasal passage indicates that
the N. fowleri trophozoite is more neurotropic as compared to aniso-
tropic in the progression of PAM; additionally, the mobility of tro-
phozoites is due to chemotaxis, not chemokinesis. This chemotactic
mobility leads to an important question of which chemical is involved
in generating the neurochemical gradient for the chemotaxis of tro-
phozoites of N. fowleri towards the brain. This selective chemotaxis
of N. fowleri trophozoites towards the neural tissue can be explained
by the fact that N. fowleri has a cell surface receptor for a specific
chemo‐attractant ligand that stimulates its mobility and prolifera-
tion. Furthermore, the chemical basis of neuro‐chemotaxis shown
by N. fowleri can be further fortified, if microfilament and actin as-
sembly are enhanced by the downstream signalling that is originated
from such a receptor (Figure 2).43
Acetylcholine acts as chemo‐attractant other than to act as
a neurotransmitter, for eukaryotic cells like smooth muscles, neu-
46
trophils and neurons. The neurological tissue of the brain such
as the frontal lobe and olfactory region are notably involved in the
secretion of acetylcholine. Recent studies have reported that olfac-
tory mucosa contains cholinergic and adrenergic nerves that are
involved in secretion of olfactory modulating chemical ligand such
as noradrenaline and acetylcholine, respectively.47 N. fowleri has a
cell surface G‐protein coupled receptor (GPCR) that is structurally
homologous to human M1 muscarinic receptor subtype (mAChR1) to
which acetylcholine binds. Acetylcholine binds to this trophozoites
receptor given in Figure 2, as a result, neurotropic chemotaxis can be
set up having upward movement across the cribriform plate.43
Several studies have suggested that the signal transduction path-
ways of N. fowleri were activated by the attachment of the amoeba
to a host cell that led to the production and release of mucosal
layer‐eroding proteases that promote proliferation and invasion of
this amoeba within the CNS.48 There are certain factors that cause
adhesion of this organism including pore‐forming proteins (Naegleria
pores), presence of carbohydrates residues in the outer surface of
the plasma membrane and glycol‐conjugates has terminal ‐L‐Fucose
and ‐D‐glucose.49
5 | N A EG LER I A FOW LER I A N D H OS T
IMMUNE RESPONSE
The pathogenicity of N. fowleri is attributed to its two principal char-
acteristics. First, N. fowleri contains a sucker‐like surface structure
called ‘food cup’ that enables this amoeba to digest brain tissue. The
formation of these food cups is carried out by Nf‐a1 and Nf‐actin,
which mediate the phagocytic activity of N. fowleri. 50 Second, N.
fowleri releases different cytolytic molecules including neurami-
nidases, acid hydrolases, phospholipases, and phospholipolytic
|
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enzymes, that in CNS cause further destruction of nerves. Moreover,
infection with N. fowleri also elicits an intense immune response,
causing destruction of the CNS and results in infection mediated
destruction. 51
Although the mechanism of pathogenesis of PAM is not well
understood, 50 it is considered that N. fowleri has evolved mecha-
nisms to cause evasion of the host immune system, which is why
it may be considered as a successful pathogen.41 Trogocytosis is a
process through which Entamoeba histolytica along with N. fowleri
causes devastation of their respective targets such as human cells.
The surface‐associated hemolytic factors are present in N. fowleri,
reported when chromate‐focusing studies were performed on the
whole‐cell lysates of N. fowleri, these factors were involved in their
pathogenicity.19 It was investigated that amoebae are trapped by
the host mucus at the initial stages of infection. The activation of
innate defence takes place in respiratory epithelial cells including
inflammation (IL‐8 and IL‐1 beta) and mucin secretion (MUC5AC)
by the production of reactive oxygen species (ROS), on N. fowleri
exposure.52 Mucin caused inhibition of cytotoxicity in vivo and in
vitro and binding of N. fowleri to cells but the inflammatory reac-
tion is induced by few N. fowleri pathogens after their penetration
into the epithelium. During later stages of infection, neutrophils and
eosinophils were observed to surround the N. fowleri. Experiments
performed on knockout mice to determine the role of inflammation
in damaging the tissues, which demonstrate that damage to the CNS
tissue caused by the host polymorphonuclear (PMN) cell lysis action
and inflammatory response.6
The presence of amoebic trophozoites in the human brain lesions
shown by histopathological examination revealed an inflammatory
infiltration of immune cells like macrophages, eosinophil, and neu-
trophils. The activated neutrophils played a prime role in the prior
stage of Naegleria infections to develop lesions. The presence of the
myeloperoxidase–H2O2–halide system in neutrophils contributes an
important role in expressing anti‐amoebic activity. This response to
N. fowleri infection is further enhanced by tumour necrosis factor
alpha (TNFα) which facilitates the adherence to N. fowleri when they
are activated with TNFα in vitro and then cause damage to amoebae.
Although there is no direct effect of TNFα on N. fowleri, the presence
of this pro‐inflammatory cytokine is paramount for neutrophils to
destroy the amoebae.41
The definitive mechanism against N. fowleri activities exerted
by neutrophils remains to be defined. Human peripheral blood was
used to address this issue, as a source to get purified PMNs.53 The
earliest cells that arrive at sites of infection in chemotactic signals
response are the PMNs. The wide variety of antimicrobial mech-
anisms can be displayed by PMNs such as degranulation, reactive
oxygen species (ROS), antimicrobial peptides, proteolytic enzymes
and reactive nitrogen species (RNS) can be displayed by PMNs and
these can rapidly accumulate in large numbers. 54 The PMN cells re-
leased neutrophil extracellular traps (NETs), they use antimicrobial
proteins localized on NET filaments for inactivating and catching the
pathogens.53 The neutrophil extracellular traps are the structures
that are composed of a backbone of mitochondrial or nuclear DNA
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204       JAHANGEER et al.
incorporated with protein and histones components of cytoplasmic
granules such as neutrophil elastase (NE), myeloperoxidase (MPO)
55
and antimicrobial peptides including lactoferrin and cathepsin G.
In vitro interaction of human PMNs with N. fowleri was performed
by Contis‐Montes de Oca et al,53 for the evaluation that either the
formation of NETs is induced by the direct contact with trophozoites
or in the presence of human antibodies immunoglobulin G (IgG). The
results of the experiment showed that N. fowleri trophozoites can in-
duce NETs formation in a time‐dependent manner with IgG antibod-
ies or without them (un‐opsonized and opsonized trophozoites) by
human PMNs, and when they were opsonized there is a significant
reduction in the number of trophozoites. 53 In addition, the presence
of DNA, myeloperoxidase, histones, and elastase enzymes have
56
also been found in the initiation of NET formation. The host de-
fence mechanism against Naegleria is also activated by macrophages
shown by collective evidence.41 The resident macrophages called
microglial cells, present in the brain have a varied array of amoebi-
cidal molecules that are secreted and provide a primary host defence
mechanism against the invasion of CNS caused by Naegleria.19 The
killing of N. fowleri through activated macrophages is induced by the
production of nitric oxide in non‐oxidative mediators such as IL‐1
and TNF‐α and in an arginine‐dependent cytolytic mechanism and
possibly other factors. The effect of TNF‐α is neither amoebistatic
nor amoebicidal, either it is alone or combined with IL‐1.6
N. fowleri can evade the host's immune defences by internalizing
the surface‐bound antibody. Although N. fowleri binding is inhibited
by secretory Immunoglobulin A (SIgA) antibodies, the antigen‐anti-
body complex is eradicated on the surface by the amoebae through
internalization of surface‐bound antibody and capping. 57 The patho-
genic function of the amoeba is associated indirectly by the secretion
of diverse proteins from the N. fowleri trophozoites, but this feature
is not fully understood. The amoeba causes the devastation of the
host cell and nervous system indirectly by secreting several pro-
teins including phospholipases, cysteine proteases, neuraminidases,
phospholipolytic enzymes, and acid hydrolases but still, no direct
evidence regarding the roles of these secreted proteins was avail-
able. 50 To determine the adhesion and invasion of host extracellular
matrix (ECM) by the Naegleria species both N. fowleri (pathogenic)
and N. lovaniensis (non‐pathogenic) were investigated and reported
that adhesion to the ECM components such as fibronectin, laminin‐1,
and collagen I by the N. fowleri is significantly higher. The spread‐out
appearance along with focal adhesion‐like structures is exhibited by
N. fowleri when it attached to the ECM substrate revealed by scan-
ning electron microscopy. The two integrin‐like proteins present in
both species revealed by western immune blotting, but a protein
with approximately 70 kDa molecular mass was found to be present
at a higher level in pathogenic species. There is co‐localization of in-
tegrin‐like proteins to the focal adhesion‐like structures indicated by
confocal microscopy. Moreover, one of the targets to prevent PAMs
is to inhibit the adhesion of N. fowleri to ECM components, and can
be decreased by anti‐integrin antibody. 58 Further, it was found that
Mp2CL5 protein played an important role in the pathogenicity of
the amoeba and only present in pathogenic N. fowleri. Moreover,
the more virulent amoebic trophozoites expressed this protein in a
higher amount as compared to less virulent trophozoites.48 N. fowleri
extracts contain protein kinase C that exhibits its activity by affect-
ing cytotoxicity and binding of host cells. N. fowleri can induce ROS
in its host and consequently cause damage to the host cell.59 There
is a cytolytic pore‐forming membrane‐bound protein called N‐PFP
and it is 66  kDa, expressed by N. fowleri, which affects membrane
integrity by depolarizing the membrane potential.6
A study conducted by Lam et al60 revealed that the degradation
of extracellular matrix (ECM) components caused by amoebae in
PAM, is mediated by matrix metalloproteinases (MMPs), including
MMP‐14 [membrane type‐1 matrix metalloproteinase (MT1‐MMP],
MMP‐2 (gelatinase A) and MMP‐9 (gelatinase B). Collectively, in
vitro study and in vivo study suggested that the invasion of the CNS
is facilitated by these MMPs. Another study conducted to evaluate
the effect of N. fowleri trophozoites on the tight junctions of Madin–
Darby canine kidney (MDCK) cell, suggesting that N. fowleri tropho-
zoites have a role in the disruption of epithelial monolayers, making
them able to penetrate the olfactory epithelium and hence cause
CNS invasion.61 The important components of the N. fowleri ESP are
the cysteine protease named N. fowleri cathepsin B‐like (nfcpb‐L)
and cathepsin B (nfcpb), the study indicated that these refolding
proteins have a principal role in host immune evasion, tissue inva-
sion, nutrient uptake, and encystment/excystment. Both rNfCP‐B
and rNfCPB‐L have shown proteolytic activity for several proteins
including IgA, IgG, IgM, fibronectin, haemoglobin, albumin and colla-
gen.62 Further, it was recently evaluated that N. fowleri excretory and
secretory proteins (NfESP) were involved in producing inflammatory
responses in BV‐2 microglial cells. The collective data suggested
that (NfESP) caused activation of BV‐2 microglial cells and produce
chemokines like IL‐1α, TNF‐α and pro‐inflammatory cytokines. The
pathological effects exerted by these NfESP involve not only direct
cytolytic damage to host neuronal cells but also the induction of pro‐
inflammatory immune responses.50 The important functions such as
adaptive responses for the survival of amoebae including protein
folding and regulation of the immune system of the host are essen-
tially accomplished by Heat‐shock protein 70 (HSP70) that were
identified in N. fowleri. Therefore, the pathogenicity of N. fowleri is
not affected by high temperature as N. lovaniensis is.7 Hence, both
contact‐dependent mechanism (food cups), as well as contact‐inde-
pendent mechanism (secretion of various proteins), are significantly
involved in evasion of the host immune response by N. fowleri.63
6 | PR I M A RY A M O E B I C
M E N I N G O E N C E PH A LITI S I N A N I M A L S
Naegleria fowleri also causes PAM in animals besides humans and
several unprompted cases of meningoencephalitis or encephalitis
have been reported due to free‐living amoeba in carnivores, horses,
ruminants and some wild animals.64 PAM lesions that have devel-
oped in cattle have close resemblance to those reported in humans.
The adjacent cerebral lesions and severe olfactory bulb, multifocal
JAHANGEER et al.
non‐suppurative lesions along with nerve neuritis, and N. fowleri
demonstration in the olfactory nerve indicated that the route
for pathogen entry in cattle was also nasal mucosa and olfactory
65
nerves.
During the summers of 1998 and 1999 brain samples of nine 10‐
to 20‐month‐old heifers had been observed having acute multifocal
necrosuppurative haemorrhagic meningoencephalitis along with the
clinical histories of severe disease of the CNS. Cerebella and olfac-
tory lobes were the parts severely affected. Lesions were also seen
in olfactory nerves, periventricular as well as in sub meningeal neu-
ropil.65 The clinical signs in specimens from six Holstein heifers in the
summer of 1999, obtained by the California Animal Health and Food
Safety Department, include facial paralysis, anorexia, ataxia, circling,
and convulsions. The identification of N. fowleri has also been ob-
served from one of the brains of infected cows and its identifica-
tion is based on sequencing of the internal transcribed spacers (ITS)
such as 5.8S rRNA genes and on the morphology.66 The other report
described one more case of a Holstein cow having fatal meningo-
encephalitis due to N. fowleri in an area of California by consuming
untreated surface water, where temperatures exceeded 42°C in the
summer.65
Further, more than 21 young heifers having neurological signs
in the California outbreaks, among these the brains of nine were
examined that demonstrated the amoebic trophozoites in all nine
brains, indicating that PAM is not a rare disease. N. fowleri‐associated
encephalomyelitis has also been reported in a cow from Costa Rica.
The available brain for diagnosis is only the formalin‐fixed brain. All
parts of the brain have severe meningoencephalitis and the lesions
contain many amoebic trophozoites. The indirect immunofluores-
cence antibody test indicated that N. fowleri specific polyclonal anti-
bodies, the amoebae get reacted.67
The other PAM case in a cross‐bred 1‐year‐old steer was re-
ported by Pimentael et al64 in Brazil with its clinical, pathological
and epidemiological findings. However, lesion distribution compared
to previously recorded cases was different. There was thickening of
the meninges, caudal and rostral calculi, cerebellum, parietal and oc-
cipital cortex and multifocal areas of malacia in the thalamus have
been observed at necropsy and histological examination revealed
amoebic trophozoites and immunohistochemistry confirmed the N.
fowleri. The animals used hot, stagnant water for drinking which was
considered as a major source of infection and the nasal route has
2,65,67
been suggested for N. fowleri infections in cattle.
There is another report that described two PAM cases in Algeria
in a ewe and in a cow from Batna, north‐eastern Algeria. After a
week of first clinical manifestations, both ruminants died. The amoe-
bae cells were indicated after staining of the cerebrospinal fluid with
May‐Grünwald‐Giemsa. The species‐specific real‐time PCR was used
to determine the presence of N. fowleri in histological tissue sections.
The source of infection was the drinking water used by the cattle,
from a deep well flowing into a small capacity irrigation basin, with
exposure to approximately 45°C ambient temperatures. No human
PAM cases have been recorded in Algeria or the Maghreba. The
meningitis cases in cattle are now significantly reported in different
|
      205
geographic locations worldwide; indicating that genus Naegleria is
rarely involved in causing meningitis in cattle. Experimentally, PAM
has been reproduced in sheep, rats and monkeys.64
7 | PR I M A RY A M O E B I C
M E N I N G O E N C E PH A LITI S A N D PA K I S TA N
Though PAM infection due to N. fowleri has been reported all around
the world, it is mostly reported in developed countries rather than
in developing countries, which might be due to more advanced diag-
nostic tools and increased awareness available in developed coun-
tries.68 More recently, PAM cases have been seen in the developing
countries affecting the lives of young men, such as in Pakistan, India,
etc. Most of the PAM cases arise due to recreational activities but in
Pakistan, apart from one case, all the PAM patients have no recrea-
tional activities like swimming, but this dreadful amoeba invades the
young men and results in death.42,69
The first PAM infected case was reported in Karachi, Pakistan
in 2008, in a 25‐year‐old man, who had a history of swimming
in a river.70 One more study confirmed two more cases affected
by PAM in Karachi, Pakistan. Both the patients use nearby water
sources for swimming. The other study reported 13 cases of pri-
mary amoebic meningoencephalitis within 17  months in Karachi,
Pakistan. All the persons had no history of recreational activities,
however, infection of these patients may be due to the performing
of regular ablution practices using tap water which might be the
source of N. fowleri due to contamination.71 Another study con-
ducted by Shariq et al16 reported one more case of a 42‐year‐old
male patient affected by PAM due to N. fowleri from Karachi. He
also had no swimming history and used fresh water for rinsing and
cleansing of the nose. He acquired this infection in September in
which the weather in Karachi was very warm. During a short pe-
riod of time, within 8  years, from 2008 to 2017, N. fowleri infec-
tion resulted in about 105 individuals' deaths in Pakistan (Figure 1,
Table 1), with the highest number of cases in Pakistan as many as
22 cases, being reported in the year 2012. Recently, seven more
cases have been reported.42,72
All the reported cases from Pakistan have been from only one
city and not even a single case has been reported from any other city
of Pakistan. The observation revealed that the prevalence of free‐
living amoebae (FLA) in Pakistan is high due to the current weather
conditions and global warming, in the coming years, this could be
dangerous for the community.3 It was reported that there are many
reasons that might be the major causes of a sudden increase in PAM
prevalence, such as contaminated water supply to the city, decrease
in rainfall, improper water chlorination and recent increases in heat
waves in Karachi. These environmental factors ultimately enhanced
the fresh water temperature that provided the ideal niches for the
thermophilic N. fowleri16 and the presence of both non‐pathogenic
and pathogenic thermo‐tolerant Naegleria species has been identi-
fied in Pakistan from different water sources including the drinking
water.73
 |
206       JAHANGEER et al.

TA B L E 1   Occurrence of PAM in Pakistan from 2008 to 2017 therapeutic techniques that included a number of drugs ranging
from antimicrobial compounds to experimental anti‐cancer drugs,
Number of Cases Year Age/Gender References
but still the mortality rate is very high. The major hurdles in the
71
2 2008 30‐y‐old male 25‐y‐
treatment of PAM are possibly due to complexities in early diagnosis
old male
100
that result in delay in initiation of effective chemotherapy and/or
11 2009 Age range: 16–64 y
across the blood–brain barrier (BBB), due to anatomy, in the penetra-
101
20 2010 Age range: NR and
tion of antimicrobial compounds.76 The ability of drugs to reach the
All are males
71
brain parenchyma successfully is a major choice, therefore, thera-
13 2011 Age range: NR and
peutic strategies against N. fowleri could be improved by searching
All are males
42 the molecules that have attractive amoebicidal effects as well as
22 2012 Age range: All are
males characteristics. Along with accurate diagnosis and initiation of rec-

3 2013 Age range: 14–40 y 42
All are males
14 2014 Age range: 14–40 y 42
All are males
13 2015 Age range: 16–56 y 42
10 males, 3
females
72
5 2016 Not Reported
72
2 2017 Not Reported
A noticeable death rate of approximately 20 deaths per year due
to PAM in Pakistan has been observed by the Aga Khan University
Hospital in Karachi, Pakistan (a leading private hospital).71 When
the possible risk factors such as swimming in fresh water were in-
vestigated, it was confusing that not even a single infected patient
had a swimming history. Even though Karachi is a cosmopolitan city,
all the infected persons strictly practice Islam, and most are young
men. Muslims offer prayer five times a day and for cleansing, they
practice ablution before every prayer. There is repeated irrigation
for cleansing of the face, mouth, arms, ears, feet, and nostrils for this
religious practice. Most of the people push water forcefully up to
the nostrils, while it is not mandatory to force water in this way for
ablution. There are enormous health benefits of practicing ablution
but only when the water supplies are without pathogenic microbes,
if the water contains N. fowleri, this can get access to the nasal mu-
cosa, invade the brain and cause this acute infection. Recently, in the
United States, the death of a Muslim male patient has been reported
while performing ablution with contaminated water.74 All these re-
ported cases suggested that rigorous ablution (as not recommended
by Islam religion) poses a risk in contracting PAM is a primary factor
in Pakistan.42
8 |  M A N AG E M E NT
The management of PAM due to N. fowleri is the major challenge
both for developed as well as developing countries. It is recom-
mended that treatment must be started promptly after the identi-
fication of amoebae in the CSF wet mount. Although, there is no
adequate recommended treatment that provides surety for survival,
different strategies are used to manage the PAM.7,75 The syner-
gistic effects of drugs can be achieved by using a combination of
ommended therapy, many other factors like an immune response of
the host, strain virulence and inoculums played an important role in
the management of this drastic disease.77 There is the utmost need
to develop anti‐PAM drugs by the pharmaceutical industry but due
to economic constraints, especially in developing countries, there is
a lack of interest in the development of such drugs, even though N.
fowleri infection is rare and results in loss of precious lives annually.
8.1 | Antimicrobial agents as anti‐Naegleria drugs
The treatment of such neglected diseases can be possible by a
cost‐effective strategy called drug `repurposing', for re‐profiling
known drugs. The sterol biosynthesis pathway is similar in free‐liv-
ing amoebae and pathogenic fungi that encouraged evaluating the
anti‐mycotic clinically approved drugs for anti‐Naegleria activity.78
The polyene antifungal drug, amphotericin B (AmpB), is the drug of
choice for the treatment of PAM and was supposed to be the foun-
dation of treatment. Amphotericin B acts on target cells by binding
to ergosterol present in the cell membrane alter membrane perme-
ability by forming pores and hence cause cell death. However, the
use of this drug alone or in combination with other drugs treated
not more than a dozen patients successfully while approximately
more than 350 cases of PAM have been reported worldwide.79,80
The number of patients treated with amphotericin B is 36 while only
three (8%) patients survived. 21
Several experiments were performed to determine the efficacy
of amphotericin in combination with other drugs because of its sev-
eral side effects. Amphotericin B has limited clinical use because
of its toxicity such as dose‐related nephrotoxicity and acute‐infu-
sion related reactions.78,81 AmpB may also cause anaemia and the
majority of patients experience fever, chills, vomiting, nausea, and
headache. The majority of the complexities related to amphoteri-
cin B may be associated with its reduced aqueous solubility which
affects clearance, compartmentalization, and dissolution.13,75 The
combination of AmpB with rifampin results in survival of most of the
patients, among those who survived, with a duration of one month
of hospitalization but this duration may vary and increased up to
2–3  months for full recovery.17 The only recorded survivor in the
United States was given miconazole and amphotericin intrathecally
and intravenously, and rifampin was given intravenously. The sur-
vivor in Mexico was given fluconazole, amphotericin and rifampin
intravenously. Conventional amphotericin is preferable as compared
JAHANGEER et al.
to the liposomal formulation as it has a lower minimum inhibitory
concentration, while CSF penetration of liposomal form is better.
Another medical treatment has also shown activity against N. fowleri
such as voriconazole, fluconazole and azithromycin.77
The study conducted by82 indicated that combined use of azi-
thromycin and amphotericin B may be effective for the treatment
of PAM as both are synergistic against the Lee strain of N. fowleri.
The effectiveness of an anti‐parasitic agent, miltefosine has been
shown in vitro against N. fowleri and other clinically important FLA.
The first successful treatment of PAM using miltefosine was con-
ducted in 2015.77 The infections caused by free‐living amoeba can
be treated with miltefosine and it is available directly from CDC, in
the United States, while a study showed permanent brain damage
75,83
on patients treated with miltefosine. Further, animal trials re-
vealed that the survival rate of experimental mice with chlorprom-
azine, miltefosine and amphotericin B were 75%, 55%, and 40%,
respectively. 81
Another in vitro study was conducted to identify an efficient,
non‐toxic, fast‐acting and water‐soluble drug‐like corifungin which
has the ability to kill both pathogenic N. fowleri and non‐pathogenic
N. gruberi effectively and has higher activity than that of amphoter-
icin B with the same mechanism of action as Amp B. The damage to
both surface and internal membranes of N. fowleri could be caused
by low concentrations of corifungin shown by ultrastructure analysis.
The analysis also showed that corifungin could target the mitochon-
dria of N. fowleri. Therefore, an orphan drug designation for cori-
fungin has been approved by the US Food and Drug Administration
(FDA) for the treatment of PAM. 84
The building blocks of plasma membranes are ergosterol, es-
sentially required for both N. fowleri and fungi proliferation. The
sterol 14‐demethylase (CYP51) is encoded by the N. fowleri ge-
nomes that have approximately 35% sequence identical to fun-
gal orthologous. The rapid death of pathogenic amoebae can be
caused by the disturbance of sterol biosynthesis. Therefore, a
study was carried out by x‐ray crystallography and biochemical
TA B L E 2   PAM survivors with different
PAM survivor
treatment regimen
26‐y‐old female
10‐y‐old boy
8‐mo‐old infant
12‐y‐old girl
8‐y‐old Hispanic
boy
40‐y‐old male
16‐y‐old boy
12‐y‐old
Caucasian girl
|
      207
studies of the target enzyme which elaborated that disruption of
sterol biosynthesis in amoebae can be caused by conazoles, the
azole antifungal drugs that compete for binding to the active site of
the sterol 14‐demethylase (CYP51), with the natural binding sub-
strate.78 The most important property while selecting an agent,
to test against the pathogen is their BBB penetration property.
Therefore, for the survival of the patients and their full neuro-
logic recovery, it is mandatory to identify amoebicidal compounds
having good BBB permeability. A study performed to identify the
central nervous system (CNS) active compounds, ebselen, BAY
11‐7082 and BAY 11‐7085 that are active against N. fowleri tropho-
zoites, have the ability to penetrate the BBB. The study indicated
that incubation of N. fowleri trophozoites with ebselen results in
loss of the nuclear membrane, in the cytoplasmic membrane al-
teration and appearance of electron‐dense granules while incuba-
tion with BAY 11‐7082 and BAY 11‐7085 cause cytoplasmic and
nuclear membranes disruption, in the cytoplasm presence of large
lipid droplets and appearance of chromatin residues and several
vesicles, has been observed. These amoebicidal compounds that
are permeable to the BBB are considered as potential new drugs
for the treatment of N. fowleri. 63
Nanoformulation technology was recently used to enhance
drug availability by conjugation with silver nanoparticles and three
drugs named Nystatin, Amphotericin B and Fluconazole were used
for the determination of amoebicidal effects, silver nanoparticles
alone, and drugs alone and drug‐conjugated silver nanoparticles
were incubated with N. fowleri. These findings revealed that silver
nanoparticle conjugation increases the efficiency of anti‐amoebic
drugs significantly.85 The current treatment for PAM patients rec-
ommended by the CDC includes combined therapy that consists of
miltefosine, an investigational anti‐cancer agent, antibiotics rifampin
and azithromycin, anti‐mycotic drugs fluconazole and amphotericin
B (AmpB) and finally for the reduction of cerebral oedema, use of
dexamethasone, an anti‐inflammatory drug.78 Table 2 shows PAM
survivors along with treatment description.
Year Treatment description References
102
2002 Rifampicin, amphotericin B, imidazole, treat-
ment was continued for 3 wk
103
2005 Intravenous (iv) dexamethasone, amphotericin
B, fluconazole, and oral rifampicin
104
2008 Amphotericin B, chloramphenicol and ri-
fampicin for 3 wk
77
2013 Amphotericin B, fluconazole, rifampin,
azithromycin, dexamethasone, miltefosine
20
2013 Amphotericin B, rifampin, fluconazole,
azithromycin, miltefosine, dexamethasone
12
2015 Amphotericin B, Metronidazole, Rifampicin
74
2016 Amphotericin, rifampin, azithromycin, flucona-
zole, dexamethasone, and miltefosine
105
2016 Amphotericin, rifampin, azithromycin, flucona-
zole, dexamethasone, and miltefosine
 |
208       JAHANGEER et al.
8.2 | Molecular based targets and vaccination
strategies to manage PAM
Vaccination strategies to manage a pathogen remain an important
therapeutic regimen and it was reported that a vaccine developed
against N. fowleri has a potential role. In 1977 John and colleagues
investigated that injecting different preparations of N. fowleri im-
munized the mice against lethal parasitemia doses. Immunization
of mice with formalized or live N. fowleri or live N. gruberi induced
a potential to fight with N. fowleri as compared to no immunized
mice and it was also reported that as compared to other routes, the
intravenous route is preferable to stimulate the immune system.
Further, N. gruberi extracts and the whole parasite is more immu-
nogenic as compared to N. fowleri extracts and fragmented parasite,
respectively.86 Thong et al revealed that the inoculation of culture
media used for amoebae growth into mice provided more immunity
against N. fowleri, especially protection that occurred in the nasal
mucosa.87 In another study, it was determined that combined ad-
ministration of Bacillus thuringiensis Cry1Ac protoxin and N. fowleri
lysate provided the prolonged survival in mice compared to the mice
only receiving N. fowleri lysate. Both mucosal IgG and IgA production
enhanced, and the level of IgG remained high even after 2 months of
88
inoculation and in the nasal lumen, IgA interaction with trophozo-
ites was also reported on comparing to the non‐immunized animal
where trophozoites penetrated into the nasal mucosa.89 Molecular
based study revealed that Cry1Ac as an adjuvant administered along
with amoebal lysate in STAT6+/+ mice has a 100% survival rate on
comparison with STAT6−/− mice who did not survive on introduc-
ing intranasal N. fowleri. The results suggested Th2‐biased immune
response which was correlated with the presence of STAT6 protein.
Further, high production of IgG1 and IL‐4 took place in the STAT6+/+
mice while the STAT6−/− mice showed IL‐12, IFN‐γ and Th1‐associ-
ated IgG2a production.90
Protein expression by introducing DNA vaccination in 1990 was
the milestone in the history of vaccine development, upon injecting
directly the plasmid DNA into myocytes to produce proteins.91 DNA
vaccine led to the production of endogenous proteins which discov-
ered the production of immunoglobulin against specific pathogens.
Further, this type of vaccination has a wide range of advantages
including simplicity, easy administration, stability, significantly im-
munogenic in nature, and the immune system elicited remained for
a long time.92 Both immune types, including humoral and cellular
types, are effectively activated by DNA vaccination and characterize
a promising strategy in human protection against various pathogens
like mycobacteria, human immunodeficiency virus and parasites.93-95
Different types of vectors such as plasmids, viral DNA are used as
vectors to carry the desired gene.96
A gene expressed in the pseudopodia called the nfa1 gene having
a molecular weight of 360 bp and encodes Nfa1 protein (13.1 kDa).
N. fowleri‐induced host cell cytotoxicity can be reduced by gene si-
lencing of nfa1 and anti‐Nfa1 antibody. As the pathogenesis of N.
fowleri is greatly influenced by the nfa1 gene, experiments were
performed to examine the effect of nfa1 DNA vaccination against
N. fowleri infection by using lentiviral vector (pCDH). The results
showed that cytokine production, production of specific IgG and IgG
subclasses (IgG1 and IgG2a) are strongly enhanced by nfa1 vaccina-
tion, and the increase in survival rate (90%) of mice infected with N.
fowleri trophozoites has also been observed. Furthermore, this study
suggested that for the treatment of N. fowleri infection, nfa1 vacci-
nation could be a new potential tool.97 Another study revealed that
protective immune responses can be obtained by immunization of
mice with rNfa1 protein. The results obtained from this study indi-
cated that for the treatment as well as vaccination against N. fowl-
eri infection, Nfa1 protein is a candidate antigen.98 Recently one
more study conducted using retroviral vector (pQCXIN) expressing
the nfa1 gene, to evaluate the effect of nfa1 vaccination against N.
fowleri infection. The significant increase in levels of IgG and IgG
subclass has been observed in DNA vaccinated mice. The cytokine
analysis showed that the production of IL‐4 and IFN γ cytokines is
induced by DNA vaccinated mice, this finding suggests a Th1/Th2
mixed type immune response. After N. fowleri infection the survival
rate is increased significantly, that is 60%, by using retroviral vector
for nfa1 vaccinated mice. Hence protective immunity is induced by
nfa1 vaccination using a retroviral vector, and it may be a proper trial
for the prevention and treatment of PAM.99
The study conducted for the inhibition of cysteine protease that
is involved in the progression of PAM. In vitro activity of cysteine
protease inhibitor called synthetic vinyl sulfone, K11777, has been
observed against cysteine protease activity in the lysate of N. fowl-
eri, along with 33 analogues with pyridylalanine, phenylalanine or
valine at the P2 position. The more effective inhibitors that inhibit
cysteine protease activity of N. fowleri are with pyridylalanine or
phenylalanine at the P2 position, with half‐maximal inhibitory con-
centration (IC50) ranging between 3 nmol/L to 6.6 μmol/L..75
9 | PR E V E NTI O N
Based on various findings now it is considered that this lethal and
acute disease is associated with water, as the causative agent of
PAM is a thermophilic amoeba, which proliferates in less chlorin-
ated water in warmer months of the year. Therefore, it's a matter of
urgency to maintain the chlorine level in the water, as all the PAM
cases are linked with the use of inadequately chlorinated water
for swimming, ablution practices and nasal rinsing using neti pots.
Public awareness is one of the major factors regarding this disease
particularly in developing countries like Pakistan. There should be
awareness campaigns and boards in schools, colleges, mosques and
at recreational places in Pakistan to make people aware of this brain‐
eating amoeba.
There are certain personal actions that might be helpful in re-
ducing the PAM risk such as use of untreated freshwater or tap
water should be avoided, use of distilled, sterile and filtered water
(use of filter having an absolute pore size of 1 μm or less than that)
for rinsing, flushing or irrigating nasal passages, or to make the ir-
rigation solution, use of previously boiled water. The irrigation
JAHANGEER et al.
device should be rinsed after each use with the previously boiled,
1
filtered, distilled and sterile water. According to the World Health
Organization (WHO), free chlorine having residual concentration
equal or more than 0.5 mg/L, at pH less than 8.0 and at 20°C, after
a contact time of at least 30  minutes is effective for chlorine dis-
infection. Throughout the distribution system, this level should be
maintained. The CDC recommends certain preventive measures to
avoid this infection, such as the use of purified and commercially
available distilled bottled water while using water for sinus rinsing.
The CDC recommends more preventions in case of absence of the
above‐mentioned options for sinus rinsing, including the use of a fil-
ter having smaller pores or one micron for water filtration or boiling
of water.13 The nasal irrigation should be practiced by using saline
solution (in purified or filtered warm water, 0.9% non‐iodized sodium
chloride and a buffering agent such as sodium bicarbonate may or
may not be added. When taking part in recreational activities like
swimming, avoid getting water up the nose, digging in or stirring
up the sediment and try to avoid water‐related activities when the
water temperature is high as in summer.1
10  | CO N C LU S I O N
Naegleria fowleri, a thermophilic flagellated amoeba known as “brain‐
eating” amoeba is the aetiological agent of primary amoebic menin-
goencephalitis (PAM) both in human as well as in animals with a
mortality rate more than 95%. PAM is a more prevalent disease in
developed countries and recently has been reported also in devel-
oping countries with warmer seasons. The most propitious environ-
ment adapted by N. fowleri to cause this infection is contaminated
warmer water. Infective trophozoites form to gain entry through
the nose, crossing the cribriform plate to reach the human brain
and cause severe destruction of the CNS, leading to brain haemor-
rhage and eventually death occurs within 3–7 days, if not diagnosed
and treated instantaneously. Moreover, delayed diagnosis and lack
of appropriate treatment strategies make this disease more lethal.
Although drugs like amphotericin Bused as combination therapy to
handle the PAM, the survival rate is still 5%. So, finally it could be
concluded that progress in diagnostic techniques, development of
anti‐parasitic drugs and vaccination might be the major fields for re-
searchers and pharmaceutical laboratories to manage the PAM at
the early stages. For this both animal as well as human trials at large
scale are required which will need technical and large financial sup-
port. Further, strategies to boost up the protective immune system,
as well as preventive measures, are the primary concerns to control
the pathogenic amoebae.
AC K N OW L E D G M E N T
We appreciate “Eman Diagnostic Laboratories, Lahore‐Pakistan
(PHC Regd # R‐14074)” and the doctors who supported us for the
data collection regarding this dreadful infectious disease.
|
      209
C O N FL I C T O F I N T E R E S T
There is no conflict of interest regarding the publication of this
article.
ORCID
Imtiaz Mahmood Tahir  https://orcid.org/0000-0003-4594-8869
Syed Muhammad Ali Shah  https://orcid.
org/0000-0003-3825-5589
Rida Zainab  https://orcid.org/0000-0002-3335-5436
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