Home Protozoa Naegleria fowleri
Naegleria fowleri PDF Print E-mail
Thursday, 14 January 2010 00:00
Authors
Andreas Nocker
Francine Marciano-Cabral

  • Causes a fatal disease of the central nervous system
  • transmitted while swimming or diving in contaminated water
  • can tolerate temperatures up to 45°C
  • strong preference for naturally warm or thermally polluted waters
  • can serve as a reservoir for pathogenic bacteria

The genus Naegleria comprises free-living amoebae that normally feed as phagotrophs on bacteria and detritus ( AWWA 2006). Out of more than 30 species of Naegleria, only one species, Naegleria fowleri, is known to cause a fatal disease of the human central nervous system (primary amoebic meningoencephalitis, PAM) ( Martinez AJ 1985; Visvesvara et al. 2007). Death occurs within 3-14 days after exposure ( Martinez AJ 1993; WHO Guidelines 2003b). This pathogen enters the human host through the nasal passages while swimming or diving in amoeba-contaminated water ( AWWA 2006). Although cases of disease are relatively rare, exposure to the pathogens might be more widespread as antibodies to Naegleria spp. are common in human sera ( Marciano-Cabral et al. 1987; Marshall et al. 1997). The N. fowleri lifecycle comprises three stages: the trophozoite, the flagellate, and a cyst stage ( Martinez AJ 1985). The motile flagellate stage serves to allow rapid spread to fresh water reservoirs when it rains before eventually reverting to the trophic stage ( Marshall et al. 1997; Warhurst DC 1985). In addition to causing a fatal human infection, Naegleria can serve as a reservoir for pathogenic bacteria ( Marciano-Cabral F 2004). Also, amoebae in water distribution systems can provide a surface for bacterial biofilm formation ( Marciano-Cabral et al. 2010).
Although its preferred environment is soil, rain and runoff water introduce N. fowleri into surface waters ( Marshall et al. 1997). Isolation has been reported for many water sources including fresh water, tap water, swimming pools, natural hot springs, ponds, lakes, rivers, bathtubs, backyard wading pools, and thermally polluted water from industrial sources like powerplant discharges and thermal effluents from industrial processes ( AWWA 2006; de Jonckheere and van de Voorde 1977; Griffin JL 1983; Marciano-Cabral et al. 2003; Martinez AJ 1993; Sheehan et al. 2003; Wellings et al. 1977, Willaert and Stevens 1976, WHO Guidelines 2003). This thermotolerant pathogen was reported to have a strong preference for naturally warm waters, or to those which are thermally polluted ( Laseke et al. 2009; Warhurst DC 1985; WHO Guidelines 2003b). It proliferates in water when the ambient temperature rises above 30°C (Visvesvara et al. 1997) and has the ability to survive high temperatures up to 45°C ( Visvesvara et al. 2007; Warhurst DC 1985). Based on ecological studies, it was hypothesized that N. fowleri might be more common in artificially thermal habitats than in naturally warm environments, where other, non-pathogenic thermophilic Naegleria species predominate ( WHO Guidelines 2003b). Infections occur typically in summer months when people use recreational waters.

Examples of studies on occurrence in water are:

  • Water from a lake in Virginia, which was simultaneously used for cooling the reactors of a nuclear power plant and for recreational activities, was tested for presence of N. fowleri using a nested PCR approach. Nine out of 16 sites sampled in summer months were reported positive. However, total amoeba counts (including N. fowleri) did not exceed 12 cells per 50 ml at any site. No correlation with water quality parameters was found. It was hypothesized that N. fowleri levels were kept low by predation of other protozoa and invertebrates, water disturbance by recreational boating activities, and the presence of bacterial or fungal toxins which were discharged into the water ( Jamerson et al. 2009). No cases of PAM have occurred at this lake.
  • 46% (n=26) lakes in Florida tested positive for pathogenic Naegleria using cultivation and indirect FA microscopy. Isolation frequency positively correlated with water temperature. In winter months, isolates could only be obtained from lake bottom samples. Amoebae population in three lakes reached one amoeba per 25 ml during the hot summer months ( Wellings et al. 1977).
  • Of 939 samples from artificially heated river water downstream of a nuclear power plant, 283 tested positive using an ELISA assay. Concentrations from 0.8-46 amoebae per liter were reported ( Reveiller et al. 2002).
  • Concentrations of N. fowleri found in cooling pond water ranged from zero to 104 per liter ( Reveiller et al. 2002).
N. fowleri cysts are killed by exposure to chlorine for 1 h at a concentration of 0.5 ppm ( De Jonckheere and van de Voorde 1976). The same holds true for N. lovaniensis, which is a close relative to N. fowleri and might serve as a safer-to-handle substitute ( Ercken et al. 2003). To achieve 100% killing of N. lovaniensis with monochloramine and peracetic acid, 3.9 and 5.3 ppm had to be used, respectively ( Ercken et al. 2003). The detection of N. fowleri by PCR in the low-pH Mallard Lake in Yellowstone national park suggested that it can tolerate acidic environments ( Sheehan et al. 2003). The pH optimum was determined to be 6.5 ( Cerva L 1978).
The cysts which form under adverse environmental conditions can survive for prolonged periods in moist environments, soil and water. Cysts that were stored at 4°C for up to 8 months and then cultured were able to cause disease in mice ( AWWA 2006). Infectivity was lost when cysts were dried, frozen or heated above 51°C ( AWWA 2006; Warhurst DC 1985).

N. fowleri preferentially grows in warm water with temperatures between 25 - 44°C ( Cabanes et al. 2001). It was reported that stagnant sections of domestic water supplies might favor growth and increase the risk of exposure ( Marciano-Cabral et al. 2003). Cases of disease occurred in Australia when houses remained unoccupied for longer periods during warm weather. Warm climates might favor growth in water supplies under these conditions ( Anderson et al. 1973; Carter RF 1972; Miller et al. 1982).

In lakes, pathogenic Naegleria are believed to survive in lake bottom sediments ( Wellings et al. 1977). Sediments provide more stable temperature conditions, which was suggested to play a role in overwintering: In a laboratory study, 50% of cysts survived for 35 days at either 4°C or 22°C at constant temperature. However, only 16% of cysts survived when held at 22°C for 8 h with a subsequent temperature shift to 4°C for 16 h over the same time period ( Wellings et al. 1977).

Significant numbers of this pathogen have been found in water layers containing cyanobacteria, eubacteria, and coliforms which serve as food sources. Also the presence of Fe2+ in the water might be beneficial as indicated by increased isolation frequency of N. fowleri from a hypolimnetic iron layer of a lake ( Kyle and Noblet 1985).

The infectious dose is unknown for humans. In mice, a clear correlation between dose and mortality was observed: 100 % of mice (n=20) died after intranasal instillation of 3,400 cells per mouse. Mortality gradually decreased to 30% (n=20) when the dose was decreased to 70 cells/mouse ( Cabanes et al. 2001). Based on this animal infection studies, a probability model estimated a risk of disease for human of 8.5 x 10-8 when swimming in water with a concentration of 10 N. fowleri per liter ( Cabanes et al. 2001). This assessment prompted French health authorities to recommended that a maximum level of 100 N. fowleri amoeba per liter should not be exceeded.

In Florida, 7 cases of disease were reported for around one billion water exposures in lake water. N. fowleri concentrations might have been as high as 25 amoebae per liter in summer months ( Wellings et al. 1977).

Due to morphological similarities, indistinguishable behavior in cell culture, and common antigenic structure, N. fowleri cannot be easily distinguished from other thermotolerant Naegleria species (especially N. lovaniensis) ( Reveiller et al. 2002).

A selection of molecular and immunologic detection methods is summarized in the following:

  • Immunological tests using polyclonal antibodies to Naegleria fowleri  have limited specificity and exhibit cross-reactivity with other Naegleria species ( Stevens et al. 1980). A species-specific monoclonal antibody, however, forms the basis for an ELISA applied to primary cultures of environmental water samples ( Reveiller et al. 2005). No cross-reactivity with non-pathogenic Naegleria was observed. The relatively low sensitivity of the assay per se of 2,000  trophozoites per ml  makes a prior cultivation step (on agar plates) necessary. The time required for cultivation is between 3-4 days. This assay is used for daily monitoring of water downstream of a nuclear power plant in France as a preventive measure. The assay has the advantage that water volumes of around 100 ml can be processed.
  • Multiplex PCR-RFLP: The assay uses a combination of ITS primers and species-specific primers. N. fowleri could be successfully distinguished from other amoeba: The presence of contaminating amoeba resulted in the amplification of the ITS sequence, but no PCR amplicon was obtained with the N. fowleri-specific primers ( Pélandakis and Pernin 2002). The ITS-RFLP variation of the assay can be used to genotype different Naegleria species and N. fowleri strains
  • Nested PCR: A nested PCR assay was developed with a detection limit of 5 pg DNA (corresponding to 5 amoebae) in 50 ml of water. Primers did not amplify DNA from non-pathogenic Naegleria species. The PCR assay also works using intact amoebae as the DNA source instead of using purified genomic DNA. Whole cell detection was also positive, when intact amobae were diluted in tapwater or river/lake water ( Reveiller et al. 2002). In an application of the method, cultivation was optionally used prior to amplification to eliminate potential PCR inhibition ( Marciano-Cabral et al. 2003).
  • qPCR and melting curve analysis: A quantitative PCR assay was developed targeting the 5.8 S rRNA gene and flanking ITS regions. The assay was validated with amoebae from different sources after cultivation on plates. Melting profiles could readily distinguish between 7 different thermophilic Naegleria species and the related Willaertia magna. The assay could detect DNA equivalent to one cell ( Robinson et al. 2006).
  • Multiplex qPCR: A triplex real-time PCR assay was developed for simultaneous detection of N. fowleri together with Acanthamoeba spp. and Balamuthia mandrillaris. The assay targets 18S rRNA genes and was validated with in vitro cultures and clinical specimens. Detection down to one amoeba per sample was reported ( Qvarnstrom et al. 2006).
  • IF-Solid-phase cytometry (ChemScan): The principle is based on an immuno-fluorescent  labeling of microorganisms after concentration by filtration on a membrane and detection of labeled cells by solid-phase cytometry. A detection limit between 200-500 cells per liter was reported and results were obtained within 3 h. Highest possible water volumes to be processed are 2-5 ml (dependent on the concentration of suspended matter) ( Pougnard et al. 2002)

Images are courtesies of Dr. Francine Marciano-Cabral.

Figure 1:

           N. fowleri co-cultured with bacteria (scanning electron micrograph)         

Pure culture of N. fowleri (scanning electron micrograph)

Figure 2:     

           A scanning electron micrograph of N. fowleri co-cultured with bacteria.             

N. fowleri co-cultured with bacteria (scanning electron micrograph).

Figure 3: 

            Transmission electron micrograph of N. fowleri trophozoites.                

N. fowleri trophozoites (transmission electron micrograph)

 

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