Primary amebic meningoencephalitis (PAM) is a rare and typically fatal infection caused by the thermophilic free-living ameba, Naegleria fowleri. In 2010, the first confirmed case of PAM acquired in Minnesota highlighted the need for improved detection and quantification methods in order to study the changing ecology of N. fowleri and to evaluate potential risk factors for increased exposure. An immunomagnetic separation (IMS) procedure and real-time PCR TaqMan assay were developed to recover and quantify N. fowleri in water and sediment samples. When one liter of lake water was seeded with N. fowleri strain CDC:V212, the method had an average recovery of 46% and detection limit of 14 amebas per liter of water. The method was then applied to sediment and water samples with unknown N. fowleri concentrations, resulting in positive direct detections by real-time PCR in 3 out of 16 samples and confirmation of N. fowleri culture in 6 of 16 samples. This study has resulted in a new method for detection and quantification of N. fowleri in water and sediment that should be a useful tool to facilitate studies of the physical, chemical, and biological factors associated with the presence and dynamics of N. fowleri in environmental systems. 1. Introduction Naegleria fowleri, a thermophilic free-living ameba found in freshwater environments, causes primary amoebic meningoencephalitis (PAM), a rare and typically fatal disease in children and young adults [1, 2]. In the USA, N. fowleri is commonly detected in warm freshwater environments such as lakes, rivers, inadequately disinfected swimming pools, geothermal waters (e.g., hot springs), thermally impacted surface water (e.g., from power plants), and water distribution systems [3–15]. While N. fowleri is generally considered to be widespread in the environment, especially in warm weather geographic areas, environmental factors are likely associated with the distribution of PAM in the USA. In recent years there has been an increase in the geographical distribution of PAM cases in the USA and in 2010, the first confirmed case of PAM was identified in Minnesota, the northernmost USA state in which this infection has been documented [9, 16]. These developments highlight the need for improved environmental detection and quantification methods in order to study the ecology of N. fowleri and to evaluate potential risk factors for increased exposure to this ameba. The most probable number (MPN) method is reliant on culture methods developed in the 1970s [17] and consists of spreading Escherichia coli over agar plates and
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