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Naegleria fowleri
Kelly Fero - ParaSite
February 26, 2010
Introduction
Naegleria fowleri is a free-living ameboflagellate that can cause primary
amebic meningoencephalitis in humans (PAM). Of the 30+ species of Naegleria
that have been isolated, only N. fowleri has been demonstrated to be pathogenic
in humans. Another species, N. australeinsis, has been proven to be pathogenic
in mice and is useful in laboratory study of Naegleria pathogenesis (De
Jonchheere, 2004). While the number of reported cases of N. fowleri infection is
small, because of the fatality of PAM (98% death rate), the amoeba and resulting
meningoencephalitis are a public health interest.
Scientific Classification
Naegleria fowleri are part of the kingdom Protista (subkingdom: Protazoa).
Naegleria are part of the same superclass (Rhizopodea) as other parasitic amoebas
including Entamoeba histolytica and Acanthamouba spp. A full taxonomic classification
is in Table 1 at right (adapted from Sawyer & Griffin, 1975).
Table 1. Taxonomic classification
Kingdom:
Subkingdom:
Phylum:
Subphylum:
Superclass:
Class:
Order:
Family:
Genus:
Species:
Protista
Protozoa
Sarcomastigophora
Sarcodina
Rhizopodia
Acarpomyxea
Schizopyrenida
Vahlkampfiidae
Naegleria
fowleri
History of Discovery
Dr.’s Fowler and Cutler first described human disease caused by ameboflagellate in Australia in 1965 (Fowler & Cutler, 1965). Their work on amebo-flagellates
was quite ground-breaking as it provided an example of how one protozoa can
effectively live both freely in the environment, and in a human host. In the years since a
total of 144 cases have been confirmed in a variety of countries (Table 2). In 1966 Dr.
Butt termed the infection resulting from N. fowleri
Primary Amebic Meningoencephalitis (PAM) in order to distinguish this
central nervous system (CNS) invasion from other secondary invasions other
amoebas such as E. histolytica can cause (Butt, 1966). An interesting
retrospective study found the likely first recorded case of PAM occurred in Ireland
in 1909 (St. Symmers, 1969).
Morphology (Martinez, 1985)
There are three distinct morphological stages in the life cycle of N. fowleri:
trophozoite, flagellate, and cyst (Figure 1A-C). The trophozoite is the infective
stage of the amoeba. They are ~10-20m long and contain a nucleus with a
large karyosome surrounded by a halo. Trophozoites reproduce by binary fission
and are motile due to round processes filled with granular cytoplasm called
lobopodia. N. fowleri is a thermophilic organism and can tolerate temperatures
up to 45C; the ideal growth temperature for trophozoites is 42C. When freeliving, trophozoites use a structure called a food-cup (Figure 1D) to ingest
bacteria and yeast – in a human host this same structure is used to ingest red
blood cells, white blood cells, and tissue. Another important structure is the
contractile vacuole. This vacuole ruptures, empties, and reforms in a rapid
process and is valuable in recognizing amebic trophozoites among other tissue
cells.
The flagellate stage is entered as a response to a change in pH or ion
concentration of the amoeba’s environment. In just minutes to a few hours
trophozoites differentiate into bi-flagellated cells. This change can be induced by
placement of trophozoites from culture into distilled water.
Additionally, in unfavorable conditions (low nutrient, crowding, cold
temperatures, desiccation), N. fowleri can form cysts. These cysts are ~8-15m
long and if they are introduced to the favorable environment of the human nasal
passages can revert to the trophozoite stage and become infective.
Figure 1: Stages of N. fowleri.
(A) trophozoite
(B) cyst
(C) Flagellate
(D) EM of food cup
Life Cycle (CDC, 2009)
The life cycle of N. fowleri can occur in a human host, or freely in an
aquatic or soil environment (Figure 2). In a warm, high nutrient, aquatic
environment the trophozoite stage predominates. This is the reproductive stage
and a trophozoite that undergoes promitosis results in two trophozoites. If pH or
ionic changes occur surrounding the organism, the trophozoite can transition to
the more mobile flagellated form. If the environment becomes depleted of
nutrients, cold, or dry the trophozoite can encyst to survive the unfavorable
conditions. Cysts and trophozoites can enter the human through nasal
passages, usually related to water activities. Trophozoites are infective, and their
penetration of the nasal mucosa and subsequent migration to the brain results in
PAM. Visualization of trophozoites in a person’s CSF or brain tissue is
considered the diagnostic stage.
Transmission/Reservoir
Naegleria fowleri have been isolated from soil, swimming pools, cooling
towers, hospital hydrothermal pools, and sewage sludge (Visvesvara et al.,
1990). Most reported infections occur after swimming in warm bodies of water.
Introduction of trophozoites to the nasal passages of humans is the first step in
Naegleria fowleri infection. There are no animal reservoirs of N. fowleri. The
bodies of water and soil contaminated with N. fowleri may be considered physical
reservoirs – as a free-living amoeba they can survive out of human hosts as long
as the conditions remain favorable.
Incubation Period
The period between initial contact with the pathogenic N. fowleri and the
onset of clinical signs and symptoms varies from 2-3 days to as long as 7-15
days. Once symptomatic, however, progression of PAM is rapid and often fatal
(Ma et al., 1990).
Pathogenesis (Ma et al., 1990)
The portal of entry of N. fowleri into the human host is the nasal cavity.
After entry, the trophozoite penetrates the nasal mucosa and migrates along
mesaxonal spaces of unmyelinated olfactory nerves terminating at the olfactory
bulb in the subarachniod space. This space is quite vascularized and is a route
of dissemination of trophozoites to other areas of the CNS. There are
histopathological characteristics of the invaded tissues – for example the
olfactory mucosa and olfactory bulb have hemorrhagic necrosis. An important
note is that trophozoites only are found in PAM lesions.
Clinical Presentation in Humans (Ma et al., 1990)
After the initial incubation period, N. fowleri infection is characterized by
abrupt onset of bifrontal or bitemporal headache, fever, nausea, vomiting (often
projective), and encephalitis. Sometimes early in the progression of disease
changes in smell (parosmia) and taste (ageusia) occur as trophozoites damage
the olfactory system. After early signs described above, progression to coma
and seizures is rapid – over a period of 3-7 days. PAM often resembles purulent
bacterial meningitis and early in its course differences cannot be distinguished.
The vast majority of PAM cases end in death (98%), on average only one week
after appearance of the first symptoms.
Infected tissue, upon close inspection has distinct macro and microscopic
morphology. Macroscopically, the cerebral hemispheres are observed to be
swollen and olfactory bulbs are necrotic and hemorrhagic. Trophozoites can be
foun in fascicles of unmeylinated axons of the olfactory nerves and in nasal
mucosa. On a smaller scale, the cortical gray matter is observed as a preferred
site of ingestion. Also, trophozoites can be identified in purulent exudates by
their prominent karyosome.
Diagnosis
Because of the incredibly quick progression of PAM, rapid diagnostics
must be developed for early detection, as the progression of the disease is so
fast. Currently much of the clinical diagnosis is based on patient history –
whether or not the patient has recently swam in warm bodies of water – along
with presenting symptoms. Final diagnostic confirmation is not achieved until
trophozoites are isolated and identified from CSF or brain tissue. While N.
fowleri do grow easily in culture, this can take multiple days – time that is
precious in an infection that will kill in a week.
Current research is, understandably, focused on development of real time
PCR diagnostic methods. One method being developed involves monitoring the
amplification process in real-time with hybridization of fluorescent labeled probes
targeting the MpC15 sequence – which is unique to N. fowleri (Madarova et al.,
2009). Another group has multiplexed three real-time PCR reactions as a
diagnostic for N. fowleri, as well as Acanthamoeba spp. And Balamuthia
mandrillaris (Qvarnstrom et. al, 2006). This could prove to be an incredibly
efficient diagnostic test.
Treatment
Currently, if N. fowleri infection is diagnosed or suspected treatment
Amphotericin B is the standard of care. Amphotericin B is a polyene compound
that disrupts selective permeability of plasma membranes. It is administered
intravaneously and is something of a ‘last resort’ drug as it has high toxicity.
While not particularly effective, every one of the 4 documented survivors of PAM
have been treated with Amphotericin B.
As there is no effective treatment for PAM, the development of a
therapeutic is an area of great research interest. Currently, much work is being
done to determine what specific to N. fowleri makes it pathogenic and if these
virulence factors can be targeted by drugs. One potential player in motility of the
amoeba is the Nfa1 protein. When Nfa1 is expressed in non-pathogenic N.
gruberi and the amoebas are co-cultured with target tissue cells, it was observed
that the protein was located on the food cup which is responsible for ingestion of
cells during feeding(Song et al., 2006). Following up that research, Nfaq gene
expression knockdown experiments were preformed using RNAinterference. In
this experiment dsRNA targeting the Nfa1 sequence was introduced and
subsequently expression levels of the gene product dramatically decreased
(Jung et al., 2008). This method could, potentially be a technique applicable for
knockdown of expression of pathogenicity factors in N. fowleri trophozoites.
Epidemiology
PAM due to Naegleria fowleri has a worldwide distribution and occurs
most frequently in tropical areas and during hot summer months. The majority of
the reported cases, 121 from 1937-2007, occurred in the United States (Primary
Amebic Meningoencephalitis, 2008). A distribution of those infections that
occurred in the U.S between 1937-1990 can be seen in Figure 3 (Visvesvara et
al., 1990). This high rate of occurance in the U.S. is likely due to under-reporting
elsewhere, and not a dramatically higher prevalence in the U.S. Most cases are
diagnosed upon autopsy, and in many countries autopsy is not standard.
Globally, there were a total of 144 cases reported through 1990 – the U.S.,
Australia, and the Czech Republic reported the majority of these (Visvesvara et
al., 1990). Major outbreaks, including one south of Richmond in Virginia, and one
in the Czech Republic, tend to be the result of swimming in a warm body of
water.
Figure 3. Distribution of PAM in the United States from 1937-1990 (Visvesvara
et al., 1990)
Public Health Prevention Strategies
Currently there are no widespread efforts for prevention because of the
low prevalence of N. fowleri infections. However, because of the fatality of the
ensuing meningoencephlitis there are efforts in research and development of
both diagnostics and treatment (see above). Additionally, a case can be made
for increased awareness of N. fowleri and its infection for more accurate
reporting.
Useful Websites
Information from the United States Center for Disease Control:
http://www.cdc.gov/ncidod/dpd/parasites/Naegleria/factsht_naegleria.htm
General information (wikipedia = user generated):
http://en.wikipedia.org/wiki/Naegleria_fowleri
eMedicine summary of N. fowleri and PAM:
http://emedicine.medscape.com/article/223910-overview
CDC Morbidity and Mortality reports from PAM cases:
http://www.cdc.gov/search.do?q=%2CNaegleria%2Cfowleri&btnG.x=0&btnG.y=0
&subset=mmwr&sort=date&oe=UTF-8&ie=UTF-8&ud=1&site=mmwr&site=mmwr
Works Cited
Butt CG (1 966) Primary amebic meningoencephalitis. N Engl J Med 274:14731476
De Jonchheere JF (2004). Molecular definition and the ubiquity of species in the
genus Naegleria. Protist 155: 89–103.
Fowler M & Carter RF (1965) Acute pyogenic meningitis probably due to
Acanthamoeba sp.: a preliminary report. Br Med J 2: 740–742.
Jung, S.Y., et al., Naegleria fowleri: nfa1 gene knock-down by double-stranded
RNAs. Exp Parasitol, 2008. 118(2): p. 208-13.
Life Cycle of Naegleria fowleri. Free-Living Amebic Infections July 20, 2009
[cited 2010 February 26]; Available from:
http://www.dpd.cdc.gov/dpdx/hTML/Frames/AF/FreeLivingAmebic/body_FreeLivingAmebic_naegleria.htm.
Ma, P., et al., Naegleria and Acanthamoeba infections: review. Rev Infect Dis,
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Madarova, L., et al., A real-time PCR diagnostic method for detection of
Naegleria fowleri. Exp Parasitol, 2009.
Martinez AJ (1985) Free-Living Amoebas: Natural History,
Prevention, Diagnosis, Pathology, and Treatment of Disease.
CRC Press, Boca Raton, Fla.
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Sawyer TK, Griffin JL (1975). A proposed new family, Acanthamoebidae, nfam.
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Serrano-Luna, J., et al., A biochemical comparison of proteases from pathogenic
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Song, K.J., et al., Naegleria fowleri: functional expression of the Nfa1 protein in
transfected Naegleria gruberi by promoter modification. Exp Parasitol, 2006.
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St. Symmers, W. C. 1969. Primary amoebic meningoencephalitis in Britain. Brit.
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Qvarnstrom, Y., et al., Multiplex real-time PCR assay for simultaneous detection
of Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri. J Clin
Microbiol, 2006. 44(10): p. 3589-95.
Visvesvara, G.S. and J.K. Stehr-Green, Epidemiology of free-living ameba
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