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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, Abstract
Pakistan Naegleria fowleri, a thermophilic flagellate amoeba known as a “brain‐eating” amoeba,
2
College of Allied Health
is the aetiological agent of a perilous and devastating waterborne disease known
Professionals, Directorate of Medical
Sciences, Government College University as primary amoebic meningoencephalitis (PAM), both in humans as well as in ani-
Faisalabad, Faisalabad, Pakistan
mals. PAM is a rare but fatal disease affecting young adults all around the world,
3
Department of Biochemistry, University of
Agriculture, Faisalabad, Pakistan
particularly in the developed world but recently reported from developing countries,
4
Department of Eastern with 95%–99% mortality rate. Swimmers and divers are at high risk of PAM as the
Medicine, Directorate of Medical warm water is the most propitious environment adapted by N. fowleri to cause this
Sciences, Government College University
Faisalabad, Faisalabad, Pakistan 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
Correspondence
Naveed Munir, Department of Biochemistry, destruction of the central nervous system (CNS). The brain damage leads to brain
Government College University Faisalabad, haemorrhage and death occurs within 3–7 days in undiagnosed cases and maltreated
Faisalabad, Pakistan.
Email: [email protected] 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 inoculums are involved in determining the time span between initial
contact with the pathogenic N. fowleri and the appearance of clinical
Naegleria fowleri is a free‐living, thermophilic, pathogenic flagellate signs and symptoms that may vary from 2 to 3 days to up to as long
amoeba belonging to the Heterolobosea class. In warmer months of as 7–15 days.16
the year, N. fowleri proliferates as it has the ability to tolerate tem- PAM is a deadly and rapidly spreading disease and many of the
peratures up to 45°C and feeds predominantly on bacteria on living cases have been reported in the USA, as described by Anser and
in natural bodies of warm freshwater, from where it has been fre- Hasan,17 where most of the people were involved in a swimming ac-
quently detected.1,2 N. fowleri is also known as amphibolic amoeba tivity. Recently PAM cases in Pakistan have been reported mostly
and there are three morphological stages of Naegleria species life in males, but not in females, because of ritual rinsing of the nose
cycle have been identified: trophozoite (10–25  mm), pear‐shaped for ablution commonly with contaminated water, although in reli-
temporary flagellate stage (10–16 mm) and cyst stage (8–20 mm).3,4 gious practice this action decreased chances of various infections.
Depending on the environmental conditions, N. fowleri can In Pakistan the incidence of PAM in males and females may vary
change its phenotype, and trophozoite is the reproductively‐active and has been reported as a 2:1 ratio, which is different from that in
stage which it exhibits under favourable conditions and it is consid- the US, where the predominance of PAM in females (63% of cases)
ered as the infective stage. It has a single nucleus and multiplies by has been observed, while the median age of this acute infection
binary fission; the nuclear membrane remains intact during this divi- is 11  years (range from 4 to 56  years).5 Most PAM cases were re-
5
sion (a process known as pro mitosis). The appropriate temperature ported during summer in the United States, though in the winter
for the best growth of this stage is 35–46°C. Trophozoites transform months, in northern states as well as in southern states with colder
to a temporary motile stage called flagellate when nutritional de- waters such as Connecticut and Pennsylvania, N. fowleri presence
ficiency occurs in the environment, but water is present, and this has been recorded but not even a single case has been found be-
stage thrives best in the temperatures rang 27–37°C. Under unfa- tween December and March. Further, more recently, PAM cases
vorable or adverse conditions, the metabolically dormant cyst stage were reported from Karachi, Pakistan.18 This review article has been
originates from the trophozoites and can survive in very low tem- compiled to summarize the possible risk factors for this infection,
peratures. Both flagellate and cyst forms of N. fowleri are non‐re- pathogenesis, diagnosis and management strategies. Further, the
productive and non‐feeding stages, while only the trophozoites can burden and challenges for Pakistan to handle this disease are also
6,7
reproduce, feed, and/or become cysts. described briefly.
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 2 | H I S TO R I C A L BAC KG RO U N D A N D
formation of food cups, chemotaxis and chemokines. 6,8
N. fowleri E PI D E M I O LO G Y O F PR I M A RY A M O E B I C
is the only species that causes a lethal brain infection called pri- M E N I N G O E N C E PH A LITI S
mary amoebic meningoencephalitis (PAM), while over 40 species
of Naegleria have been identified. Though PAM is rare, it is a fatal The first case of PAM was reported in Florida, USA, in 1962. The
9,10
human disease with a mortality rate of 95%–97%. The death name Naegleria fowleri was given to it after the discovery of the
occurs within approximately seven days, as it is a lethal, necrotiz- disease primary amoebic meningoencephalitis (PAM) in Australia
ing, fulminant, and haemorrhagic meningoencephalitis.11 PAM oc- in 1965 by Malcom Fowler of the Adelaide Children's Hospital,
curs significantly in immunologically strong individuals as well as in Australia, and R. F. Carter, who first described this disease. The
healthy children and young adults, having recent exposure to recre- name “primary amoebic meningoencephalitis” was given to it in
ational freshwater.12 PAM is a waterborne disease, so most cases are 1966 by Butt and then in 1968 by Carter to differentiate it from
associated with diving and swimming activities in less chlorinated the rare brain invasion caused by Entamoeba histolytica. PAM cases
pools, polluted canals and spas or during leisure sports such as water have been reported from 15 countries all over the globe, excluding
skiing in contaminated environmental water sources, and the use of Antarctica,12,19 numbering only a few hundred and are mostly from
3
neti pots for nasal cleansing and ablution. Europe, Australia, and the United States, as well as from some Asian
This acute infection is developed by the entry of N. fowleri countries.6 Though the number of annually reported infections re-
through nasal cavity when water is forced or splashed into the nose. mained stable (0–8), recent changes in the epidemiology of PAM are
N. fowleri causes destruction of neurons and explains why this is of great concern. A PAM case from the northern state of Minnesota
also known as the “brain‐eating amoeba”; this term shows that the was reported for the first time in 2010, then in 2011 and 2012 fol-
enzymes and toxins of this parasite are typically involved in the de- lowed by additional cases from Indiana, Minnesota, and Kansas; this
struction (eating‐up) of the brain.13,14 PAM is characterized by similar led to increased concerns about an expanding geographic range of
signs and symptoms to those of viral or bacterial meningitis including this infection caused by the heat‐loving, potentially climate‐sensitive
fever, headache, stiff neck, vomiting, anorexia, seizures, ultimately pathogen. 20
death typically occurs within 3–7 days after the appearance of these The number of PAM patients reported from 1937 to 2013 was
signs and symptoms.15 The virulence of the strain and the size of the 142, in the United States. A vast study has revealed that from 1962
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to 2014, throughout the world approximately 260 cases have been ponds, freshwater lakes, and rivers while urban zones include con-
observed which include: 132 cases from the United States, 19 in taminated and uncontaminated water sources, drinking water dis-
Australia, 17 cases from Pakistan, 16 cases from the Czech Republic, tribution system (DWDS) through pipe wall biofilms, 29 hotel and
11 from India, 9 in Mexico, 9 from New Zealand, 7 from Venezuela, domestic swimming pools, recreational fountains, hospitals, geo-
5 cases each from Thailand and Belgium, 4 from Nigeria, 2 from the thermal heated water, contaminated drinking water and water parks,
United Kingdom, and 1 case only from each of Namibia, Iran, Costa dental unit waterlines (DUWLs), and nasal flooding using tap water.
Rica, New Guinea, South Africa and Madagascar. It was also reported The number of N. fowleri is greater in sites having increased water
that only 11 patients survived out of these 260 reported cases.17,21 temperature that is above 28ºC.30 Hence, environmental monitor-
According to recent studies 16 cases of PAM have been reported ing by using accurate and fast screening is essentially required as
in India, only four patients survived out of these, while three patients risk prevention for discrimination between other free‐living amoeba
had no water contact, so it becomes doubtful that the infection was (FLA) and N. fowleri in water samples.31,32
due to N. fowleri. The majority of other patients in India used ground- The crucial points while fighting N. fowleri includes its detec-
water, puddles, or ponds for bathing or swimming. N. fowleri pres- tion and dispersal mode as both non‐pathogenic and pathogenic
ence has been confirmed in pond water, swimming pools and sewage species present in the environment. There are different methods to
canals of India.7,22 It was also reported in other countries like China, specifically identify N. fowleri such as immunological or molecular,
Hong Kong, Zhejiang, Zambia (Figure 1). 23,24 other recent methods are also included visually detecting N. fowl-
According to the US Center for Disease Control and Prevention eri. 27,33 Molecular based techniques like polymerase chain reaction
(CDC), the number of PAM cases in the United States from 1962 (PCR) are more sensitive, rapid and specific as compared to culture
to 2015 was 138. Recently, in Asian countries, an increase in the and microscopic techniques.32 The direct quantification and de-
23
number of PAM cases has been investigated. With respect to the tection of microorganisms in environmental samples can be done
occurrence of PAM, in the USA 143 cases have been reported from particularly by quantitative PCR (qPCR), without requiring cultural
1962 to 2016, 139 of which were fatal. 25 Around 440 cases are re- isolation and such real‐time PCR‐based diagnostic methods might
corded worldwide. 26,27 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
3 | H A B ITAT A N D D I AG N OS I S O F the identification of N. fowleri isolates. Later for the identification
N .  fow l e r i of N. fowleri, isoenzymes and antibodies were used. The typing of
different strains of N. fowleri and Naegleria spp. was done by using
Naegleria fowleri, being a thermophilic protist, preferentially in- electrophoretic karyotyping (KE) and restriction‐fragment‐length
habits warm water including lakes in the tropics and hot springs in polymorphism (RFLP). Experimental work shows that from Europe,
temperate zones. 28 However, N. fowleri trophozoites or cysts have Australia, USA, and New Zealand, the N. fowleri strains differ in iso-
been classified depending upon its habitat in the most varied en- enzymes, KE as well as in RFLP.8
vironments into two categories, natural habitats, and urban zones. The one nucleotide base pair difference in the 5.8 S rDNA and
Natural habitats include hot springs and warm aquatic conditions, 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,
F I G U R E 1   Worldwide reported cases of PAM due to Naegleria proteomics and fingerprinting approach, by using a database search
fowleri until 2017 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 may be significant in disease progression such as the trophozoites
I N FEC TI O N ability to attach to the nasal mucosa, a chemotactic response to
components of nerve cell, an increased locomotion rate.41,42
The acute infection in the human body is developed when water Clinical symptoms such as a change in the smell sense and re-
containing N. fowleri is forcefully inhaled into the upper nasal pas- spiratory infection signify involvement of olfactory epithelium and
sages during bathing, swimming, or other recreational activities.18 neural tissue invasion. 2 The signs and symptoms shown by patients
This infection occurs firstly by attachment of amoeba to the nasal affected by PAM do not include any symptom of nasal inflammation
mucosa, which then moves along the olfactory nerve and reaches such as nasal pain, bleeding, tenderness at the nose bridge, sneezing,
the olfactory bulbs through the cribriform plate within the CNS and/or persistent rhinorrhea, before developing signs of PAM such
(Figure 2). Furthermore, there is a possibility that the risk of PAM as meningitis. Moreover, damage and destruction of the olfactory
is higher among those children and young adults that have more bulb, olfactory mucosa and adjacent areas of the brain are mostly
porous cribriform plates.11,39 Research has revealed that infection observed after postmortem findings, while similar destruction in the
cannot be initiated by drinking contaminated water.40 Another study region of non‐olfactory mucosa of the nasal cavity is not observed.43
indicated that this amoeba can only infect a person through the ol- One study indicated that N. fowleri favours the frontal lobe as com-
factory route, not by the oral route. There are certain events that pared to the parietal lobe. As N. fowleri adopted the nasal route for
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entry, showing that N. fowleri probably has a closed association with enzymes, that in CNS cause further destruction of nerves. Moreover,
frontal lobe, this is because the olfactory bulb has anatomical prox- infection with N. fowleri also elicits an intense immune response,
imity to the frontal lobe; in the nasal passage it is terminal to the causing destruction of the CNS and results in infection mediated
olfactory neuro‐epithelium, and hence traverses the cribriform plate destruction. 51
44,45
to the brain described diagrammatically in Figure 2. Although the mechanism of pathogenesis of PAM is not well
The absence of any damage in the nasal passage indicates that understood, 50 it is considered that N. fowleri has evolved mecha-
the N. fowleri trophozoite is more neurotropic as compared to aniso- nisms to cause evasion of the host immune system, which is why
tropic in the progression of PAM; additionally, the mobility of tro- it may be considered as a successful pathogen.41 Trogocytosis is a
phozoites is due to chemotaxis, not chemokinesis. This chemotactic process through which Entamoeba histolytica along with N. fowleri
mobility leads to an important question of which chemical is involved causes devastation of their respective targets such as human cells.
in generating the neurochemical gradient for the chemotaxis of tro- The surface‐associated hemolytic factors are present in N. fowleri,
phozoites of N. fowleri towards the brain. This selective chemotaxis reported when chromate‐focusing studies were performed on the
of N. fowleri trophozoites towards the neural tissue can be explained whole‐cell lysates of N. fowleri, these factors were involved in their
by the fact that N. fowleri has a cell surface receptor for a specific pathogenicity.19 It was investigated that amoebae are trapped by
chemo‐attractant ligand that stimulates its mobility and prolifera- the host mucus at the initial stages of infection. The activation of
tion. Furthermore, the chemical basis of neuro‐chemotaxis shown innate defence takes place in respiratory epithelial cells including
by N. fowleri can be further fortified, if microfilament and actin as- inflammation (IL‐8 and IL‐1 beta) and mucin secretion (MUC5AC)
sembly are enhanced by the downstream signalling that is originated by the production of reactive oxygen species (ROS), on N. fowleri
from such a receptor (Figure 2).43 exposure.52 Mucin caused inhibition of cytotoxicity in vivo and in
Acetylcholine acts as chemo‐attractant other than to act as vitro and binding of N. fowleri to cells but the inflammatory reac-
a neurotransmitter, for eukaryotic cells like smooth muscles, neu- tion is induced by few N. fowleri pathogens after their penetration
46
trophils and neurons. The neurological tissue of the brain such into the epithelium. During later stages of infection, neutrophils and
as the frontal lobe and olfactory region are notably involved in the eosinophils were observed to surround the N. fowleri. Experiments
secretion of acetylcholine. Recent studies have reported that olfac- performed on knockout mice to determine the role of inflammation
tory mucosa contains cholinergic and adrenergic nerves that are in damaging the tissues, which demonstrate that damage to the CNS
involved in secretion of olfactory modulating chemical ligand such tissue caused by the host polymorphonuclear (PMN) cell lysis action
as noradrenaline and acetylcholine, respectively.47 N. fowleri has a and inflammatory response.6
cell surface G‐protein coupled receptor (GPCR) that is structurally The presence of amoebic trophozoites in the human brain lesions
homologous to human M1 muscarinic receptor subtype (mAChR1) to shown by histopathological examination revealed an inflammatory
which acetylcholine binds. Acetylcholine binds to this trophozoites infiltration of immune cells like macrophages, eosinophil, and neu-
receptor given in Figure 2, as a result, neurotropic chemotaxis can be trophils. The activated neutrophils played a prime role in the prior
set up having upward movement across the cribriform plate.43 stage of Naegleria infections to develop lesions. The presence of the
Several studies have suggested that the signal transduction path- myeloperoxidase–H2O2–halide system in neutrophils contributes an
ways of N. fowleri were activated by the attachment of the amoeba important role in expressing anti‐amoebic activity. This response to
to a host cell that led to the production and release of mucosal N. fowleri infection is further enhanced by tumour necrosis factor
layer‐eroding proteases that promote proliferation and invasion of alpha (TNFα) which facilitates the adherence to N. fowleri when they
this amoeba within the CNS.48 There are certain factors that cause are activated with TNFα in vitro and then cause damage to amoebae.
adhesion of this organism including pore‐forming proteins (Naegleria Although there is no direct effect of TNFα on N. fowleri, the presence
pores), presence of carbohydrates residues in the outer surface of of this pro‐inflammatory cytokine is paramount for neutrophils to
the plasma membrane and glycol‐conjugates has terminal ‐L‐Fucose destroy the amoebae.41
and ‐D‐glucose.49 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
5 | N A EG LER I A FOW LER I A N D H OS T earliest cells that arrive at sites of infection in chemotactic signals
IMMUNE RESPONSE response are the PMNs. The wide variety of antimicrobial mech-
anisms can be displayed by PMNs such as degranulation, reactive
The pathogenicity of N. fowleri is attributed to its two principal char- oxygen species (ROS), antimicrobial peptides, proteolytic enzymes
acteristics. First, N. fowleri contains a sucker‐like surface structure and reactive nitrogen species (RNS) can be displayed by PMNs and
called ‘food cup’ that enables this amoeba to digest brain tissue. The these can rapidly accumulate in large numbers. 54 The PMN cells re-
formation of these food cups is carried out by Nf‐a1 and Nf‐actin, leased neutrophil extracellular traps (NETs), they use antimicrobial
which mediate the phagocytic activity of N. fowleri. 50 Second, N. proteins localized on NET filaments for inactivating and catching the
fowleri releases different cytolytic molecules including neurami- pathogens.53 The neutrophil extracellular traps are the structures
nidases, acid hydrolases, phospholipases, and phospholipolytic 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 the more virulent amoebic trophozoites expressed this protein in a
granules such as neutrophil elastase (NE), myeloperoxidase (MPO) higher amount as compared to less virulent trophozoites.48 N. fowleri
55
and antimicrobial peptides including lactoferrin and cathepsin G. extracts contain protein kinase C that exhibits its activity by affect-
In vitro interaction of human PMNs with N. fowleri was performed ing cytotoxicity and binding of host cells. N. fowleri can induce ROS
by Contis‐Montes de Oca et al,53 for the evaluation that either the in its host and consequently cause damage to the host cell.59 There
formation of NETs is induced by the direct contact with trophozoites is a cytolytic pore‐forming membrane‐bound protein called N‐PFP
or in the presence of human antibodies immunoglobulin G (IgG). The and it is 66  kDa, expressed by N. fowleri, which affects membrane
results of the experiment showed that N. fowleri trophozoites can in- integrity by depolarizing the membrane potential.6
duce NETs formation in a time‐dependent manner with IgG antibod- A study conducted by Lam et al60 revealed that the degradation
ies or without them (un‐opsonized and opsonized trophozoites) by of extracellular matrix (ECM) components caused by amoebae in
human PMNs, and when they were opsonized there is a significant PAM, is mediated by matrix metalloproteinases (MMPs), including
reduction in the number of trophozoites. 53 In addition, the presence MMP‐14 [membrane type‐1 matrix metalloproteinase (MT1‐MMP],
of DNA, myeloperoxidase, histones, and elastase enzymes have MMP‐2 (gelatinase A) and MMP‐9 (gelatinase B). Collectively, in
56
also been found in the initiation of NET formation. The host de- vitro study and in vivo study suggested that the invasion of the CNS
fence mechanism against Naegleria is also activated by macrophages is facilitated by these MMPs. Another study conducted to evaluate
shown by collective evidence.41 The resident macrophages called the effect of N. fowleri trophozoites on the tight junctions of Madin–
microglial cells, present in the brain have a varied array of amoebi- Darby canine kidney (MDCK) cell, suggesting that N. fowleri tropho-
cidal molecules that are secreted and provide a primary host defence zoites have a role in the disruption of epithelial monolayers, making
mechanism against the invasion of CNS caused by Naegleria.19 The them able to penetrate the olfactory epithelium and hence cause
killing of N. fowleri through activated macrophages is induced by the CNS invasion.61 The important components of the N. fowleri ESP are
production of nitric oxide in non‐oxidative mediators such as IL‐1 the cysteine protease named N. fowleri cathepsin B‐like (nfcpb‐L)
and TNF‐α and in an arginine‐dependent cytolytic mechanism and and cathepsin B (nfcpb), the study indicated that these refolding
possibly other factors. The effect of TNF‐α is neither amoebistatic proteins have a principal role in host immune evasion, tissue inva-
nor amoebicidal, either it is alone or combined with IL‐1.6 sion, nutrient uptake, and encystment/excystment. Both rNfCP‐B
N. fowleri can evade the host's immune defences by internalizing and rNfCPB‐L have shown proteolytic activity for several proteins
the surface‐bound antibody. Although N. fowleri binding is inhibited including IgA, IgG, IgM, fibronectin, haemoglobin, albumin and colla-
by secretory Immunoglobulin A (SIgA) antibodies, the antigen‐anti- gen.62 Further, it was recently evaluated that N. fowleri excretory and
body complex is eradicated on the surface by the amoebae through secretory proteins (NfESP) were involved in producing inflammatory
internalization of surface‐bound antibody and capping. 57 The patho- responses in BV‐2 microglial cells. The collective data suggested
genic function of the amoeba is associated indirectly by the secretion that (NfESP) caused activation of BV‐2 microglial cells and produce
of diverse proteins from the N. fowleri trophozoites, but this feature chemokines like IL‐1α, TNF‐α and pro‐inflammatory cytokines. The
is not fully understood. The amoeba causes the devastation of the pathological effects exerted by these NfESP involve not only direct
host cell and nervous system indirectly by secreting several pro- cytolytic damage to host neuronal cells but also the induction of pro‐
teins including phospholipases, cysteine proteases, neuraminidases, inflammatory immune responses.50 The important functions such as
phospholipolytic enzymes, and acid hydrolases but still, no direct adaptive responses for the survival of amoebae including protein
evidence regarding the roles of these secreted proteins was avail- folding and regulation of the immune system of the host are essen-
able. 50 To determine the adhesion and invasion of host extracellular tially accomplished by Heat‐shock protein 70 (HSP70) that were
matrix (ECM) by the Naegleria species both N. fowleri (pathogenic) identified in N. fowleri. Therefore, the pathogenicity of N. fowleri is
and N. lovaniensis (non‐pathogenic) were investigated and reported not affected by high temperature as N. lovaniensis is.7 Hence, both
that adhesion to the ECM components such as fibronectin, laminin‐1, contact‐dependent mechanism (food cups), as well as contact‐inde-
and collagen I by the N. fowleri is significantly higher. The spread‐out pendent mechanism (secretion of various proteins), are significantly
appearance along with focal adhesion‐like structures is exhibited by involved in evasion of the host immune response by N. fowleri.63
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 6 | PR I M A RY A M O E B I C
with approximately 70 kDa molecular mass was found to be present M E N I N G O E N C E PH A LITI S I N A N I M A L S
at a higher level in pathogenic species. There is co‐localization of in-
tegrin‐like proteins to the focal adhesion‐like structures indicated by Naegleria fowleri also causes PAM in animals besides humans and
confocal microscopy. Moreover, one of the targets to prevent PAMs several unprompted cases of meningoencephalitis or encephalitis
is to inhibit the adhesion of N. fowleri to ECM components, and can have been reported due to free‐living amoeba in carnivores, horses,
be decreased by anti‐integrin antibody. 58 Further, it was found that ruminants and some wild animals.64 PAM lesions that have devel-
Mp2CL5 protein played an important role in the pathogenicity of oped in cattle have close resemblance to those reported in humans.
the amoeba and only present in pathogenic N. fowleri. Moreover, The adjacent cerebral lesions and severe olfactory bulb, multifocal
JAHANGEER et al. |
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non‐suppurative lesions along with nerve neuritis, and N. fowleri geographic locations worldwide; indicating that genus Naegleria is
demonstration in the olfactory nerve indicated that the route rarely involved in causing meningitis in cattle. Experimentally, PAM
for pathogen entry in cattle was also nasal mucosa and olfactory has been reproduced in sheep, rats and monkeys.64
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 7 | PR I M A RY A M O E B I C
necrosuppurative haemorrhagic meningoencephalitis along with the M E N I N G O E N C E PH A LITI S A N D PA K I S TA N
clinical histories of severe disease of the CNS. Cerebella and olfac-
tory lobes were the parts severely affected. Lesions were also seen Though PAM infection due to N. fowleri has been reported all around
in olfactory nerves, periventricular as well as in sub meningeal neu- the world, it is mostly reported in developed countries rather than
ropil.65 The clinical signs in specimens from six Holstein heifers in the in developing countries, which might be due to more advanced diag-
summer of 1999, obtained by the California Animal Health and Food nostic tools and increased awareness available in developed coun-
Safety Department, include facial paralysis, anorexia, ataxia, circling, tries.68 More recently, PAM cases have been seen in the developing
and convulsions. The identification of N. fowleri has also been ob- countries affecting the lives of young men, such as in Pakistan, India,
served from one of the brains of infected cows and its identifica- etc. Most of the PAM cases arise due to recreational activities but in
tion is based on sequencing of the internal transcribed spacers (ITS) Pakistan, apart from one case, all the PAM patients have no recrea-
such as 5.8S rRNA genes and on the morphology.66 The other report tional activities like swimming, but this dreadful amoeba invades the
described one more case of a Holstein cow having fatal meningo- young men and results in death.42,69
encephalitis due to N. fowleri in an area of California by consuming The first PAM infected case was reported in Karachi, Pakistan
untreated surface water, where temperatures exceeded 42°C in the in 2008, in a 25‐year‐old man, who had a history of swimming
summer.65 in a river.70 One more study confirmed two more cases affected
Further, more than 21 young heifers having neurological signs by PAM in Karachi, Pakistan. Both the patients use nearby water
in the California outbreaks, among these the brains of nine were sources for swimming. The other study reported 13 cases of pri-
examined that demonstrated the amoebic trophozoites in all nine mary amoebic meningoencephalitis within 17  months in Karachi,
brains, indicating that PAM is not a rare disease. N. fowleri‐associated Pakistan. All the persons had no history of recreational activities,
encephalomyelitis has also been reported in a cow from Costa Rica. however, infection of these patients may be due to the performing
The available brain for diagnosis is only the formalin‐fixed brain. All of regular ablution practices using tap water which might be the
parts of the brain have severe meningoencephalitis and the lesions source of N. fowleri due to contamination.71 Another study con-
contain many amoebic trophozoites. The indirect immunofluores- ducted by Shariq et al16 reported one more case of a 42‐year‐old
cence antibody test indicated that N. fowleri specific polyclonal anti- male patient affected by PAM due to N. fowleri from Karachi. He
bodies, the amoebae get reacted.67 also had no swimming history and used fresh water for rinsing and
The other PAM case in a cross‐bred 1‐year‐old steer was re- cleansing of the nose. He acquired this infection in September in
ported by Pimentael et al64 in Brazil with its clinical, pathological which the weather in Karachi was very warm. During a short pe-
and epidemiological findings. However, lesion distribution compared riod of time, within 8  years, from 2008 to 2017, N. fowleri infec-
to previously recorded cases was different. There was thickening of tion resulted in about 105 individuals' deaths in Pakistan (Figure 1,
the meninges, caudal and rostral calculi, cerebellum, parietal and oc- Table 1), with the highest number of cases in Pakistan as many as
cipital cortex and multifocal areas of malacia in the thalamus have 22 cases, being reported in the year 2012. Recently, seven more
been observed at necropsy and histological examination revealed cases have been reported.42,72
amoebic trophozoites and immunohistochemistry confirmed the N. All the reported cases from Pakistan have been from only one
fowleri. The animals used hot, stagnant water for drinking which was city and not even a single case has been reported from any other city
considered as a major source of infection and the nasal route has of Pakistan. The observation revealed that the prevalence of free‐
2,65,67
been suggested for N. fowleri infections in cattle. living amoebae (FLA) in Pakistan is high due to the current weather
There is another report that described two PAM cases in Algeria conditions and global warming, in the coming years, this could be
in a ewe and in a cow from Batna, north‐eastern Algeria. After a dangerous for the community.3 It was reported that there are many
week of first clinical manifestations, both ruminants died. The amoe- reasons that might be the major causes of a sudden increase in PAM
bae cells were indicated after staining of the cerebrospinal fluid with prevalence, such as contaminated water supply to the city, decrease
May‐Grünwald‐Giemsa. The species‐specific real‐time PCR was used in rainfall, improper water chlorination and recent increases in heat
to determine the presence of N. fowleri in histological tissue sections. waves in Karachi. These environmental factors ultimately enhanced
The source of infection was the drinking water used by the cattle, the fresh water temperature that provided the ideal niches for the
from a deep well flowing into a small capacity irrigation basin, with thermophilic N. fowleri16 and the presence of both non‐pathogenic
exposure to approximately 45°C ambient temperatures. No human and pathogenic thermo‐tolerant Naegleria species has been identi-
PAM cases have been recorded in Algeria or the Maghreba. The fied in Pakistan from different water sources including the drinking
meningitis cases in cattle are now significantly reported in different 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 ommended therapy, many other factors like an immune response of
All are males the host, strain virulence and inoculums played an important role in
14 2014 Age range: 14–40 y 42 the management of this drastic disease.77 There is the utmost need
All are males to develop anti‐PAM drugs by the pharmaceutical industry but due
13 2015 Age range: 16–56 y 42 to economic constraints, especially in developing countries, there is
10 males, 3 a lack of interest in the development of such drugs, even though N.
females
fowleri infection is rare and results in loss of precious lives annually.
72
5 2016 Not Reported
72
2 2017 Not Reported
8.1 | Antimicrobial agents as anti‐Naegleria drugs
A noticeable death rate of approximately 20 deaths per year due The treatment of such neglected diseases can be possible by a
to PAM in Pakistan has been observed by the Aga Khan University cost‐effective strategy called drug `repurposing', for re‐profiling
Hospital in Karachi, Pakistan (a leading private hospital).71 When known drugs. The sterol biosynthesis pathway is similar in free‐liv-
the possible risk factors such as swimming in fresh water were in- ing amoebae and pathogenic fungi that encouraged evaluating the
vestigated, it was confusing that not even a single infected patient anti‐mycotic clinically approved drugs for anti‐Naegleria activity.78
had a swimming history. Even though Karachi is a cosmopolitan city, The polyene antifungal drug, amphotericin B (AmpB), is the drug of
all the infected persons strictly practice Islam, and most are young choice for the treatment of PAM and was supposed to be the foun-
men. Muslims offer prayer five times a day and for cleansing, they dation of treatment. Amphotericin B acts on target cells by binding
practice ablution before every prayer. There is repeated irrigation to ergosterol present in the cell membrane alter membrane perme-
for cleansing of the face, mouth, arms, ears, feet, and nostrils for this ability by forming pores and hence cause cell death. However, the
religious practice. Most of the people push water forcefully up to use of this drug alone or in combination with other drugs treated
the nostrils, while it is not mandatory to force water in this way for not more than a dozen patients successfully while approximately
ablution. There are enormous health benefits of practicing ablution more than 350 cases of PAM have been reported worldwide.79,80
but only when the water supplies are without pathogenic microbes, The number of patients treated with amphotericin B is 36 while only
if the water contains N. fowleri, this can get access to the nasal mu- three (8%) patients survived. 21
cosa, invade the brain and cause this acute infection. Recently, in the Several experiments were performed to determine the efficacy
United States, the death of a Muslim male patient has been reported of amphotericin in combination with other drugs because of its sev-
while performing ablution with contaminated water.74 All these re- eral side effects. Amphotericin B has limited clinical use because
ported cases suggested that rigorous ablution (as not recommended of its toxicity such as dose‐related nephrotoxicity and acute‐infu-
by Islam religion) poses a risk in contracting PAM is a primary factor sion related reactions.78,81 AmpB may also cause anaemia and the
in Pakistan.42 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
8 |  M A N AG E M E NT affects clearance, compartmentalization, and dissolution.13,75 The
combination of AmpB with rifampin results in survival of most of the
The management of PAM due to N. fowleri is the major challenge patients, among those who survived, with a duration of one month
both for developed as well as developing countries. It is recom- of hospitalization but this duration may vary and increased up to
mended that treatment must be started promptly after the identi- 2–3  months for full recovery.17 The only recorded survivor in the
fication of amoebae in the CSF wet mount. Although, there is no United States was given miconazole and amphotericin intrathecally
adequate recommended treatment that provides surety for survival, and intravenously, and rifampin was given intravenously. The sur-
different strategies are used to manage the PAM.7,75 The syner- vivor in Mexico was given fluconazole, amphotericin and rifampin
gistic effects of drugs can be achieved by using a combination of intravenously. Conventional amphotericin is preferable as compared
JAHANGEER et al. |
      207

to the liposomal formulation as it has a lower minimum inhibitory studies of the target enzyme which elaborated that disruption of
concentration, while CSF penetration of liposomal form is better. sterol biosynthesis in amoebae can be caused by conazoles, the
Another medical treatment has also shown activity against N. fowleri azole antifungal drugs that compete for binding to the active site of
such as voriconazole, fluconazole and azithromycin.77 the sterol 14‐demethylase (CYP51), with the natural binding sub-
The study conducted by82 indicated that combined use of azi- strate.78 The most important property while selecting an agent,
thromycin and amphotericin B may be effective for the treatment to test against the pathogen is their BBB penetration property.
of PAM as both are synergistic against the Lee strain of N. fowleri. Therefore, for the survival of the patients and their full neuro-
The effectiveness of an anti‐parasitic agent, miltefosine has been logic recovery, it is mandatory to identify amoebicidal compounds
shown in vitro against N. fowleri and other clinically important FLA. having good BBB permeability. A study performed to identify the
The first successful treatment of PAM using miltefosine was con- central nervous system (CNS) active compounds, ebselen, BAY
ducted in 2015.77 The infections caused by free‐living amoeba can 11‐7082 and BAY 11‐7085 that are active against N. fowleri tropho-
be treated with miltefosine and it is available directly from CDC, in zoites, have the ability to penetrate the BBB. The study indicated
the United States, while a study showed permanent brain damage that incubation of N. fowleri trophozoites with ebselen results in
75,83
on patients treated with miltefosine. Further, animal trials re- loss of the nuclear membrane, in the cytoplasmic membrane al-
vealed that the survival rate of experimental mice with chlorprom- teration and appearance of electron‐dense granules while incuba-
azine, miltefosine and amphotericin B were 75%, 55%, and 40%, tion with BAY 11‐7082 and BAY 11‐7085 cause cytoplasmic and
respectively. 81 nuclear membranes disruption, in the cytoplasm presence of large
Another in vitro study was conducted to identify an efficient, lipid droplets and appearance of chromatin residues and several
non‐toxic, fast‐acting and water‐soluble drug‐like corifungin which vesicles, has been observed. These amoebicidal compounds that
has the ability to kill both pathogenic N. fowleri and non‐pathogenic are permeable to the BBB are considered as potential new drugs
N. gruberi effectively and has higher activity than that of amphoter- for the treatment of N. fowleri. 63
icin B with the same mechanism of action as Amp B. The damage to Nanoformulation technology was recently used to enhance
both surface and internal membranes of N. fowleri could be caused drug availability by conjugation with silver nanoparticles and three
by low concentrations of corifungin shown by ultrastructure analysis. drugs named Nystatin, Amphotericin B and Fluconazole were used
The analysis also showed that corifungin could target the mitochon- for the determination of amoebicidal effects, silver nanoparticles
dria of N. fowleri. Therefore, an orphan drug designation for cori- alone, and drugs alone and drug‐conjugated silver nanoparticles
fungin has been approved by the US Food and Drug Administration were incubated with N. fowleri. These findings revealed that silver
(FDA) for the treatment of PAM. 84 nanoparticle conjugation increases the efficiency of anti‐amoebic
The building blocks of plasma membranes are ergosterol, es- drugs significantly.85 The current treatment for PAM patients rec-
sentially required for both N. fowleri and fungi proliferation. The ommended by the CDC includes combined therapy that consists of
sterol 14‐demethylase (CYP51) is encoded by the N. fowleri ge- miltefosine, an investigational anti‐cancer agent, antibiotics rifampin
nomes that have approximately 35% sequence identical to fun- and azithromycin, anti‐mycotic drugs fluconazole and amphotericin
gal orthologous. The rapid death of pathogenic amoebae can be B (AmpB) and finally for the reduction of cerebral oedema, use of
caused by the disturbance of sterol biosynthesis. Therefore, a dexamethasone, an anti‐inflammatory drug.78 Table 2 shows PAM
study was carried out by x‐ray crystallography and biochemical survivors along with treatment description.

TA B L E 2   PAM survivors with different

PAM survivor Year Treatment description References
treatment regimen
102
26‐y‐old female 2002 Rifampicin, amphotericin B, imidazole, treat-
ment was continued for 3 wk
103
10‐y‐old boy 2005 Intravenous (iv) dexamethasone, amphotericin
B, fluconazole, and oral rifampicin
104
8‐mo‐old infant 2008 Amphotericin B, chloramphenicol and ri-
fampicin for 3 wk
77
12‐y‐old girl 2013 Amphotericin B, fluconazole, rifampin,
azithromycin, dexamethasone, miltefosine
20
8‐y‐old Hispanic 2013 Amphotericin B, rifampin, fluconazole,
boy azithromycin, miltefosine, dexamethasone
12
40‐y‐old male 2015 Amphotericin B, Metronidazole, Rifampicin
74
16‐y‐old boy 2016 Amphotericin, rifampin, azithromycin, flucona-
zole, dexamethasone, and miltefosine
105
12‐y‐old 2016 Amphotericin, rifampin, azithromycin, flucona-
Caucasian girl zole, dexamethasone, and miltefosine
 |
208       JAHANGEER et al.

N. fowleri infection by using lentiviral vector (pCDH). The results

8.2 | Molecular based targets and vaccination
showed that cytokine production, production of specific IgG and IgG
strategies to manage PAM
subclasses (IgG1 and IgG2a) are strongly enhanced by nfa1 vaccina-
Vaccination strategies to manage a pathogen remain an important tion, and the increase in survival rate (90%) of mice infected with N.
therapeutic regimen and it was reported that a vaccine developed fowleri trophozoites has also been observed. Furthermore, this study
against N. fowleri has a potential role. In 1977 John and colleagues suggested that for the treatment of N. fowleri infection, nfa1 vacci-
investigated that injecting different preparations of N. fowleri im- nation could be a new potential tool.97 Another study revealed that
munized the mice against lethal parasitemia doses. Immunization protective immune responses can be obtained by immunization of
of mice with formalized or live N. fowleri or live N. gruberi induced mice with rNfa1 protein. The results obtained from this study indi-
a potential to fight with N. fowleri as compared to no immunized cated that for the treatment as well as vaccination against N. fowl-
mice and it was also reported that as compared to other routes, the eri infection, Nfa1 protein is a candidate antigen.98 Recently one
intravenous route is preferable to stimulate the immune system. more study conducted using retroviral vector (pQCXIN) expressing
Further, N. gruberi extracts and the whole parasite is more immu- the nfa1 gene, to evaluate the effect of nfa1 vaccination against N.
nogenic as compared to N. fowleri extracts and fragmented parasite, fowleri infection. The significant increase in levels of IgG and IgG
respectively.86 Thong et al revealed that the inoculation of culture subclass has been observed in DNA vaccinated mice. The cytokine
media used for amoebae growth into mice provided more immunity analysis showed that the production of IL‐4 and IFN γ cytokines is
against N. fowleri, especially protection that occurred in the nasal induced by DNA vaccinated mice, this finding suggests a Th1/Th2
mucosa.87 In another study, it was determined that combined ad- mixed type immune response. After N. fowleri infection the survival
ministration of Bacillus thuringiensis Cry1Ac protoxin and N. fowleri rate is increased significantly, that is 60%, by using retroviral vector
lysate provided the prolonged survival in mice compared to the mice for nfa1 vaccinated mice. Hence protective immunity is induced by
only receiving N. fowleri lysate. Both mucosal IgG and IgA production nfa1 vaccination using a retroviral vector, and it may be a proper trial
enhanced, and the level of IgG remained high even after 2 months of for the prevention and treatment of PAM.99
88
inoculation and in the nasal lumen, IgA interaction with trophozo- The study conducted for the inhibition of cysteine protease that
ites was also reported on comparing to the non‐immunized animal is involved in the progression of PAM. In vitro activity of cysteine
where trophozoites penetrated into the nasal mucosa.89 Molecular protease inhibitor called synthetic vinyl sulfone, K11777, has been
based study revealed that Cry1Ac as an adjuvant administered along observed against cysteine protease activity in the lysate of N. fowl-
with amoebal lysate in STAT6+/+ mice has a 100% survival rate on eri, along with 33 analogues with pyridylalanine, phenylalanine or
comparison with STAT6−/− mice who did not survive on introduc- valine at the P2 position. The more effective inhibitors that inhibit
ing intranasal N. fowleri. The results suggested Th2‐biased immune cysteine protease activity of N. fowleri are with pyridylalanine or
response which was correlated with the presence of STAT6 protein. phenylalanine at the P2 position, with half‐maximal inhibitory con-
Further, high production of IgG1 and IL‐4 took place in the STAT6+/+ centration (IC50) ranging between 3 nmol/L to 6.6 μmol/L..75
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 9 | PR E V E NTI O N
the milestone in the history of vaccine development, upon injecting
directly the plasmid DNA into myocytes to produce proteins.91 DNA Based on various findings now it is considered that this lethal and
vaccine led to the production of endogenous proteins which discov- acute disease is associated with water, as the causative agent of
ered the production of immunoglobulin against specific pathogens. PAM is a thermophilic amoeba, which proliferates in less chlorin-
Further, this type of vaccination has a wide range of advantages ated water in warmer months of the year. Therefore, it's a matter of
including simplicity, easy administration, stability, significantly im- urgency to maintain the chlorine level in the water, as all the PAM
munogenic in nature, and the immune system elicited remained for cases are linked with the use of inadequately chlorinated water
a long time.92 Both immune types, including humoral and cellular for swimming, ablution practices and nasal rinsing using neti pots.
types, are effectively activated by DNA vaccination and characterize Public awareness is one of the major factors regarding this disease
a promising strategy in human protection against various pathogens particularly in developing countries like Pakistan. There should be
like mycobacteria, human immunodeficiency virus and parasites.93-95 awareness campaigns and boards in schools, colleges, mosques and
Different types of vectors such as plasmids, viral DNA are used as at recreational places in Pakistan to make people aware of this brain‐
vectors to carry the desired gene.96 eating amoeba.
A gene expressed in the pseudopodia called the nfa1 gene having There are certain personal actions that might be helpful in re-
a molecular weight of 360 bp and encodes Nfa1 protein (13.1 kDa). ducing the PAM risk such as use of untreated freshwater or tap
N. fowleri‐induced host cell cytotoxicity can be reduced by gene si- water should be avoided, use of distilled, sterile and filtered water
lencing of nfa1 and anti‐Nfa1 antibody. As the pathogenesis of N. (use of filter having an absolute pore size of 1 μm or less than that)
fowleri is greatly influenced by the nfa1 gene, experiments were for rinsing, flushing or irrigating nasal passages, or to make the ir-
performed to examine the effect of nfa1 DNA vaccination against rigation solution, use of previously boiled water. The irrigation
JAHANGEER et al. |
      209

device should be rinsed after each use with the previously boiled, C O N FL I C T O F I N T E R E S T
1
filtered, distilled and sterile water. According to the World Health
There is no conflict of interest regarding the publication of this
Organization (WHO), free chlorine having residual concentration
article.
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 ORCID
maintained. The CDC recommends certain preventive measures to
Imtiaz Mahmood Tahir  https://orcid.org/0000-0003-4594-8869
avoid this infection, such as the use of purified and commercially
available distilled bottled water while using water for sinus rinsing. Syed Muhammad Ali Shah  https://orcid.

The CDC recommends more preventions in case of absence of the org/0000-0003-3825-5589

above‐mentioned options for sinus rinsing, including the use of a fil- Rida Zainab  https://orcid.org/0000-0002-3335-5436
ter having smaller pores or one micron for water filtration or boiling
of water.13 The nasal irrigation should be practiced by using saline
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