Improved Method for the Detection and Quantification ofNaegleria fowleriin Water and Sediment Using Immunomagnetic Separation and Real-Time PCR

Abstract

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 ofN. fowleriand to evaluate potential risk factors for increased exposure. An immunomagnetic separation (IMS) procedure and real-time PCR TaqMan assay were developed to recover and quantifyN. fowleriin water and sediment samples. When one liter of lake water was seeded withN. fowleristrain 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 unknownN. fowlericoncentrations, resulting in positive direct detections by real-time PCR in 3 out of 16 samples and confirmation ofN. fowlericulture in 6 of 16 samples. This study has resulted in a new method for detection and quantification ofN. fowleriin 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 ofN. fowleriin environmental systems.