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 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.

Methodological approaches for monitoring opportunistic pathogens in premise plumbing: A review

“…are thermophiles (Ithoi et al, 2011; Marciano-Cabral et al, 2010; Tyndall et al, 1989. Mull et al, 2013). Identities of amoeba isolates are subsequently determined according to their morphology or using molecular methods (e.g., PCR) (Garcia et al, 2013).…”

“…These assays generally target the 18S rDNA gene (Qvarnstrom et al, 2006), 5.8S gene and internal transcribed spacer (ITS) regions (Mull et al, 2013; Puzon et al, 2009) or the MP2Cl5 sequence (Behets et al, 2007a; Madarova et al, 2010). Puzon developed and applied a q-PCR assay for N. fowleri monitoring in water and biofilm samples, demonstrating higher sensitivity relative to culture methods in terms of spiked cell detection in biofilm and water (i.e., 5 cells in 250 ml water or biofilm) (Puzon et al, 2009).…”

“…Filtration, centrifugation and immuno-magnetic separation (IMS) are three common concentration methods. The first two are non-specific approaches widely used for microbial analysis, whereas IMS is able to select target microbes from background microorganisms by using antibody-coated magnetic media (Allegra et al, 2011; Bedrina et al, 2013; Mull et al, 2013). …”

Assessment of Water Quality in Roof-Harvested Rainwater Barrels in Greater Philadelphia

“…Acanthamoeba spp., Naegleria fowleri, Legionella spp., L. pneumophila, M. avium, M. intracellulare, Pseudomonas aeruginosa and C. jejuni were quantified using previously published probe-based qPCR assays [40][41][42][43][44][45]. The detailed methods previously used by the authors were replicated in this study and the full description is available elsewhere [6,13].…”

Primary Amebic Meningoencephalitis: What Have We Learned in the Last 5 Years?

“…Recently, molecular techniques based on polymerase chain reaction (PCR) and isothermal DNA amplification have been developed for the specific identification of N. fowleri in clinical and environmental samples [25-33]. Both multicopy mitochondrial 5.8S and 18S rRNA genes and internal transcribed spacers (ITS), as well as single copy genomic DNA have been used to develop these PCR assays.…”