Naegleria fowleri Kelly Fero ParaSite February 26, 2010 Introduction

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:	Protista Protozoa
Phylum: Subphylum:	Sarcomastigophora Sarcodina
Superclass:	Rhizopodia
Class:	Acarpomyxea
Order:	Schizopyrenida
Family:	Vahlkampfiidae
Genus:	Naegleria
Species:	fowleri

History of Discovery

Dr.’s Fowler and Cutler first described human disease caused by amebo-flagellate 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 free-living, 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 274:1473-1476
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/A-F/FreeLivingAmebic/body_FreeLivingAmebic_naegleria.htm.
Ma, P., et al., Naegleria and Acanthamoeba infections: review. Rev Infect Dis, 1990. 12(3): p. 490-513.
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.
Primary Amebic Meningoencephalitis --Arizona, Florida, and Texas, 2007. JAMA, 2008. 300(2): p. 161-163.
Sawyer TK, Griffin JL (1975). A proposed new family, Acanthamoebidae, nfam. (order A moebida) for certain cyst-forming filose amoebae. Trans Am Microsc Soc 94: 93-8.
Serrano-Luna, J., et al., A biochemical comparison of proteases from pathogenic naegleria fowleri and non-pathogenic Naegleria gruberi. J Eukaryot Microbiol, 2007. 54(5): p. 411-7.
Song, K.J., et al., Naegleria fowleri: functional expression of the Nfa1 protein in transfected Naegleria gruberi by promoter modification. Exp Parasitol, 2006. 112(2): p. 115-20.
St. Symmers, W. C. 1969. Primary amoebic meningoencephalitis in Britain. Brit. Med. J., 4449-454.

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 infections. J Protozool, 1990. 37(4): p. 25S-33S.