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Detection of free-living pathogenic amoebae

Research partner:
Institute of Parasitology, Faculty of Veterinary Medicine and Faculty of Medicine, University of Berne
http://www.vetmed.unibe.ch/ipa/

Background

Free-living amoebae are parasites, which infect humans and can lead to serious illness and even death. Three genera - Naegleria, Acanthamoeba und Balamuthia - are classified as human pathogenic agents. Naegleria fowleri and Balamuthia mandrillaris in particular are considered the most dangerous and therefore can be considered as potential bioweapons.

For many years, there was no institute in Switzerland working with these highly pathogenic parasites. One of the reasons may be that a biosafety level 3 laboratory facility is required.
However, our research partner possesses the necessary expertise and infrastructure to establish diagnostic systems and suitable reference material for the detection and basic study of pathogenic free-living amoebae.

Naegleria fowleri

This amoeba can be found worldwide (particularly in the United States and Australia) and can cause primary amoebic meningoencephalitis (PAM).
Infection occurs from bathing or diving in fresh water (warm stagnant pools or lakes, as well as swimming pools and whirlpools).

The amoeba enters through the nose and travels along the olfactory tract to the brain. Two to seven days after infection, acute ulcerous encephalitis occurs, accompanied by a high fever, vomiting, headaches and neck stiffness. Patients fall into a coma and most die within a week. To date there are only a few documented cases of survival, due to the patient receiving prompt treatment (amphotericin B, micanol, rifampicin).

PAM predominantly affects children and young adults in good health. Given that this amoeba occurs commonly in the environment and that many young people in endemic areas have been shown to have antibodies to Naegleria, most cases of infection are probably latent.

Naegleria fowleri trophozoites
Naegleria fowleri trophozoites
(cultivated from cerebral fluid)		 Naegleria fowleri trophozoites
Naegleria fowleri trophozoite
(in spinal fluid)

Picture source: http://www.dpd.cdc.gov/DPDx/HTML/ImageLibrary/FreeLivingAmebic_il.htm

Acanthamoeba and Balamuthia

Acanthamoeba trophozoite (scanning electron microscopy)
Acanthamoeba trophozoite (scanning electron microscopy)
Picture source: Institute for Parasitology, Berne

These amoebae probably occur throughout the world and are found in the soil, water, sand, dust and the air. Balamuthia causes granulomatous amoebic encephalitis (GAE), while Acanthamoeba primarily leads to inflammation of the cornea (acanthamoebiasis of the eye).

There are several recognised infection routes: skin lesions, droplet infection through the nasal mucous membrane (GAE) or eye keratitis from contaminated contact lenses. The incubation period can vary from between two weeks and several months, and the onset of the disease is gradual. Typical symptoms of GAE are memory loss, bouts of cramps or fever and headaches. Patients die of encephalitis within days or months; GAE is often only diagnosed post mortem. This disease mainly strikes people with an already impaired immune system.

Amoebic keratitis of the eye often goes unrecognised at the beginning, but can be treated through surgical intervention and the topical application of micanol and propamidin. The illness can be prevented by wearers taking proper care of their contact lenses.

Research aim

Establishing in vitro cultures and in vivo model systems of pathogenic free-living amoebae for the clinical diagnosis and study of pathogenesis.

Creation and validation of diagnostic PCR probes, as well as their use to understand the current epidemiological situation in Switzerland.

Identification of the pathogenic factors of Naegleria fowleri by analysing both gene and protein expression patterns with the aid of organotypic brain cultures.

Conclusions

An important diagnostic loophole can be overcome thanks to the identification and characterisation of free-living pathogenic amoebae.

Thanks to the expertise and requisite specialised laboratory facilities, we have succeeded in cultivating free-living amoebae in vitro and developing a mouse model for Naegleria fowleri within a short period of time.

Furthermore, specific diagnostic instruments (PCR, immune serums) for free-living amoebae could be generated/developed and validated.

Future results

These methods can be put to use in future work to identify pathogenic factors. This will help to make diagnoses more reliable, to understand the epidemiological situation in Switzerland and to estimate future risk potential.

Literature

Szenasi, Z; Endo T; Yagita K; Nagy E (1998): Isolation, identification and increasing importance of free-living amoebae causing human disease. J Med Microbiol; 47: 5-16

Sparagano, O; Drouet, E; Denoyel, G; Pernin, P; Ruchaud-Sparagano; MH (1994): Differentation of Naegleria fowleri from other species of Naegleria using monoclonal antibodies and the polymerase chain reaction. Trans R Soc Trop Med Hyg; 88: 119-120

Kilvington, S; Beeching, J (1995): Development of a PCR for identification of Naegleria fowleri from the environment. Appl Environ Microbiol; 61: 3764-3767

Mathers, WD; Nelson, SE, Lane, JL; Wilson, ME; Allen, RC; Folberg, R (2000): Confirmation of confocal microscopy diagnosis of Acanthamoeba keratitis using polymerase chain reaction analysis. Arch Ophtalmol 118: 178-183

Pelandakis, M and Pernin P (2002): Use of multiplex PCR and PCR restriction enzyme analysis for detection of the variability in the free-living amoeba Naegleria in the environment. Appl Environ Microbiol; 68: 2061-2065

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