Diagnosis & Detection

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Primary amebic meningoencephalitis (PAM) is a serious infection and inflammation of the brain caused by Naegleria fowleri 1, 2. The disease is diagnosed using specific laboratory tests available in only a few laboratories in the United States. Because of the rarity of the infection and difficulty in initial detection, about 75% of diagnoses are made after the death of the patient.

PAM and Naegleria fowleri infection can be diagnosed in the laboratory by detecting 3:

  • Naegleria fowleri organisms in cerebrospinal fluid (CSF), biopsy, or tissue specimens, or
  • Naegleria fowleri nucleic acid in CSF, biopsy, or tissue specimens, or
  • Naegleria fowleri antigen in CSF, biopsy, or tissue specimens.

Test Methods

The motile amebae can often be seen moving rapidly under a microscope when looking at a fresh sample of CSF. The amebae can also be stained with a variety of stains, such as Giemsa-Wright or a modified trichrome stain, for identification 1, 2.

References
  1. Visvesvara GS. Amebic meningoencephalitides and keratitis: challenges in diagnosis and treatment.external icon Curr Opin Infect Dis. 2010;23(6):590-4.
  2. da Rocha-Azevedo B, Tanowitz HB, Marciano-Cabral F. Diagnosis of infections caused by pathogenic free-living amoebae.external icon Interdiscip Perspect Infect Dis. 2009;2009:251406.

A specific antibody to Naegleria fowleri can be used in conjunction with another antibody that deposits a chemical (immunohistochemistry [IHC]) or glows under specific types of light (indirect immunofluorescence [IIF]) to directly stain the amebic antigens in tissue 1, 2.

References
  1. Visvesvara GS. Amebic meningoencephalitides and keratitis: challenges in diagnosis and treatment.external icon Curr Opin Infect Dis. 2010;23(6):590-4.
  2. da Rocha-Azevedo B, Tanowitz HB, Marciano-Cabral F. Diagnosis of infections caused by pathogenic free-living amoebae.external icon Interdiscip Perspect Infect Dis. 2009;2009:251406.

Specific molecular tools can amplify DNA from the amebae in CSF or tissue to specifically identify if the amebae are present. Looking at strains or subtypes of Naegleria fowleri can be done, but little is known about the natural populations in the environment, which makes it difficult to interpret what the findings mean 1-3.

References
  1. Qvarnstrom Y, Visvesvara GS, Sriram R, da Silva AJ. Multiplex real-time PCR assay for simultaneous detection of Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri.external icon J Clin Microbiol. 2006;44(10):3589-95.
  2. Robinson BS, Monis PT, Dobson PJ. Rapid, sensitive, and discriminating identification of Naegleria spp. by real-time PCR and melting-curve analysis. external iconAppl Environ Microbiol. 2006;72(9):5857-63.
  3. Marciano-Cabral F, MacLean R, Mensah A, LaPat-Polasko L. Identification of Naegleria fowleri in domestic water sources by nested PCR.external icon Appl Environ Microbiol. 2003;69(10):5864-9.

The amebae can be grown in culture to increase the likelihood of detecting the ameba by direct visualization or PCR 1. The sample is added to a growth plate covered in bacteria that can serve as a food source for Naegleria fowleri. The initial screening is accomplished by incubating the plate at a higher temperature (108°F/42°C) that kills most free-living amebae, while selecting for heat-loving (thermophilic) amebae, such as Naegleria fowleri or other amebae. This initial screen shows up as tracks made by an ameba as it moves across the plate eating the bacteria. If there are no amebae on the plate grown at the higher temperature, then Naegleria fowleri is not present. If heat-loving (thermophilic) amebae are present on the plate grown at the higher temperature, then these amebae undergo further specific testing to determine whether Naegleria fowleri is present since other free-living amebae can also be heat-loving (thermophilic). (NOTE: Amebae 2-4, including thermophilic amebae 5 other than Naegleria fowleri, can be common in water systems but none of these other amebae cause primary amebic meningoencephalitis [PAM]).

References
  1. Visvesvara GS. Parasite Culture: Acanthamoeba and Naegleria spp. In: Garcia LS, editor. Clinical Microbiology Procedures Handbook. 3rd ed. Washington, DC: ASM Press; 2010.
  2. Stockman LJ, Wright CJ, Visvesvara GS, Fields BS, Beach MJ. Prevalence of Acanthamoeba spp. and other free-living amoebae in household water, Ohio, USA–1990-1992.external icon Parasitol Res. 2011;108(3):621-7.
  3. Seal D, Stapleton F, Dart J. Possible environmental sources of Acanthamoeba spp. in contact lens wearers.external icon Br J Ophthalmol. 1992;76(7):424–7.
  4. Shoff ME, Rogerson A, Kessler K, Schatz S, Seal DV. Prevalence of Acanthamoeba and other naked amoebae in South Florida domestic water.external icon J Water Health. 2008;6(1):99–104.
  5. Rohr U, Weber S, Michel R, Selenka F, Wilhelm M. Comparison of free-living amoebae in hot water systems of hospitals with isolates from moist sanitary areas by identifying genera and determining temperature tolerance.external icon Appl Environ Microbiol. 1998 May;64(5):1822-4.

Water samples can be collected, concentrated, and put into culture to grow and select for Naegleria fowleri. Samples can be tested using the culture or molecular methods described above 1, 2.

References
  1. Streby A, Mull BJ, Levy K, Hill VR. Comparison of real-time PCR methods for the detection of Naegleria fowleri in surface water and sediment.external icon Parasitol Res. 2015;114(5):1739-46.
  2. Mull BJ, Jothikumar N, Hill VR. Improved method for the detection and quantification of Naegleria fowleri in water and sediment using immunomagnetic separation and real-time PCR.external icon J Parasitol Res. 2013;doi:10.1155/2013/608367.

References
  1. Marciano-Cabral F, Cabral G. The immune response to Naegleria fowleri amebae and pathogenesis of infection.external icon FEMS Immunol Med Microbiol. 2007;51:243-59.
  2. Visvesvara GS. Free-living amebae as opportunistic agents of human disease.external icon J Neuroparasitol. 2010;1.
  3. Council for State and Territorial Epidemiologists (CSTE). Case definitions for non-notifiable infections caused by free-living amebae (Naegleria fowleri, Balamuthia mandrillaris, and Acanthamoeba spp.) pdf icon[PDF – 10 pages].external icon Infectious Disease Committee. 2012.