Four genotypes of N. fowleri, identified as CDC:V212, CAMP, CDC:V515, and ATCC 30462, were grown axenically at 42°C in modified Nelson's medium (Schuster 2002 [25]).

Hartmannella vermiformis ATCC 50237, Naegleria australiensis ATCC 30958, Naegleria dunnebackei ATCC PRA-166, and Naegleria jadini ATCC 30900 were grown axenically at 37°C in modified PYNFH medium (ATCC 1034). After growth medium was removed, trophozoites were harvested in 1 mL of WB saline [26] and gently dislodged using a cell scraper. Echinamoeba exundans ATCC 50171, Naegleria clarki ATCC 30544, Naegleria gruberi ATCC 30877, Naegleria italica ATCC PRA-153, Naegleria lovaniensis ATCC 30811, Naegleria lovaniensis ATCC 30467, Tetramitus jugosus ATCC 30703, Vahlkampfia inornata ATCC 30965, and Vahlkampfia lobospinosa ATCC 30298 were grown at 37°C on nonnutrient agar (NNA) plates spread with Escherichia coli (ATCC 11775). Trophozoites or cysts were harvested in 1 mL of WB saline by gently scraping the surface of the agar plate with a cell scraper. Trophozoites were harvested 1-2 days after passing the culture to a new flask or plate, whereas cultures were left on NNA plates for 14 days to allow for all amebas to form cysts. To create a mixture of trophozoites and cysts, cultures were harvested after 4-5 days. All ameba stock concentrations were determined by four counts on a Thoma hemocytometer using 400x total magnification on a standard light microscope. After an appropriate dilution, this suspension was added to the water sample to obtain the desired seed level for all experiments.

IMS recovery experiments were performed with both amended deionized (DI) water and Georgia (GA) lake water. Deionized water was amended with 0.01 M phosphate buffered saline (PBS) to produce water samples having a conductivity of 100 μS/cm. To ensure there were no amebas in the lake water used for experiments, the water was filtered through 2.7 μm cellulose acetate filter. IMS specificity experiments were only conducted in the filtered lake water. Unfiltered GA lake water was used in the whole method comparison experiments; therefore a negative nonseeded control sample was taken through the whole procedure to ensure there was no N. fowleri present prior to seeding.

Dynabeads Biotin Binder (Invitrogen number 110.47) was precoated with Biotin-labeled anti-N. fowleri monoclonal Antibody Nf-5D12 [27] (Indicia Biotechnology, Oullins, France) at a concentration of 2 μg of biotinylated antibodies per 50 μL of Dynabeads and used within 2 weeks. The mixture was allowed to incubate for 60 min with gentle rotation and then placed on the magnet for 2 min and washed with buffer containing 0.01 M phosphate buffered saline pH 7.4, 0.1% bovine serum albumin, and 2 mM EDTA, to remove any unbound antibodies. To each water sample not exceeding 10 mL in volume, 50 μL of freshly vortexed bead-antibody mixture was added and allowed to incubate on the rotator for 30 min. A Leighton tube was placed on the Dynal MPC-6 magnet for 3 min; after the supernatant was discarded the bead pellet was resuspended in 1 mL of buffer, and the separated particles then transferred to a 1.5 mL centrifuge tube. The tube was placed on the Dynal MPC-S magnet for 2 min, supernatant was discarded again, and finally bead pellet was resuspended by vortexing in 100 μL of buffer.

The four genotypes of N. fowleri used in this study were seeded individually in both cyst and trophozoite forms at a level of 1 × 10⁵ amebas in 5 mL of amended DI water and filtered lake water. The seeded samples were then subjected to IMS in 6 replicate experiments. In addition, a mixture of 1 × 10⁵ cyst (53 ± 18%) and trophozoite (47 ± 18%) forms of 8 Naegleria spp., and 5 non-Naegleria amebas was seeded separately into 5 mL of filtered surface water in 3 replicate experiments. After the IMS procedure, the recovered amebas were counted microscopically using the hemocytometer as previously described to determine recovery efficiency of the method. One-way analysis of variance was used to test for significant differences between the mean recovery efficiencies and ameba species, ameba stage, ameba genotype, and water type using JMP v10. An alpha value of 0.05 was used for all statistical tests.

Nucleic acid extraction was performed on half of the IMS concentrate and the entire harvested cultured sample by a previously reported procedure using a noncommercial lysis buffer containing 4.5 M guanidinium isothiocyanate and bead beating using acid washed zirconium oxide beads [28].

The following TaqMan assay primers and probe sequences were selected for testing: (JBVF, 5′-AGG TAC TTA CGT TAG AGT GCT AGT-3′), (JBVR, 5′-ATG GGA CAA TCC GGT TTT CTC A-3′) and the (FAM-) labeled probe (JBVP, 5′-FAM-AC GCC CTA GCT GGT TAT GCC GGA TT-BHQ1-3′) and were evaluated for melting temperature (T_m), secondary structure and complementarity. The relative positions of the forward primer, reverse primer and probe are 166–189, 269–290 and 239–263 respectively based on GenBank accession number AJ132020. The primers amplify a 123 bp segment of the ITS region. Reactions were carried out in a 50 μL final reaction mixture using the QuantiTect probe PCR kit (Qiagen, Carlsbad, CA, USA), 5 μL of template DNA, 250 nM of the forward and reverse oligonucleotide primers, 100 nM of FAM-labeled probe, 1.0 μL of 50x nonacetylated BSA (Sigma-Aldrich St. Louis, MO, USA), and 2.5 μL of 20x T4 Gene 32 Protein (New England Biolabs, Ipswitch, MA). In each experiment, N. fowleri CDC:V212 DNA transcripts were included as a positive control and PCR-grade water was used as a negative control. All samples and dilutions were tested in duplicate on an ABI 7500 Real-time PCR Detection System (Life Technologies Corp, Carlsbad, CA, USA). Cycling conditions were (i) 95°C for 15 min (activation of Taq DNA polymerase) and (ii) 45 cycles of 95°C for 15 seconds and 63°C for 33 seconds. Fluorescence was measured at the end of each PCR cycle. A sample was considered positive when duplicate tests resulted in an average cycle threshold (CT) value <41.0. The four genotypes of N. fowleri, 8 Naegleria spp., and 5 non-Naegleria amebas previously described were tested in triplicate to determine specificity of the TaqMan assay. The DNA amount from each isolate culture used in specificity testing was equivalent to 1000 amebas/reaction; determined by hemocytometer count. A standard curve for the real-time PCR assay was tested in triplicate and prepared using a tenfold dilution series of DNA extracted from a hemocytometer-titered stock of N. fowleri CDC:V212 in DI water.

A cyst (38 ± 20%) and trophozoite (62 ± 20%) mixture of N. fowleri (CDC: V212) was added to 1 L of unaltered surface water samples in four replicate experiments. Samples were processed in parallel procedures, one with the IMS procedure and one without (the control). Each 1 L sample was concentrated by centrifuging two, 500 mL volumes in a Jouan 415 centrifuge at 1,500 × g for 15 min. After centrifugation, the supernatant was carefully removed, the pellet was resuspended, and the centrifuge tube was rinsed with WB saline. For the experimental samples, the pellet was further concentrated using the IMS procedure and the final 100 μL volume was split into three aliquots: 40 μL for agar culture, 40 μL for DNA extraction and real-time qPCR, and 20 μL for direct counting on a microscope. The control samples pellets were also split into aliquots: 1 mL for agar culture and 750 μL for DNA extraction and real-time qPCR. Agar culture plates were spread with E. coli prior to sample addition and then incubated for 7 days at 42°C. The entire culture plate was then harvested in 750 μL of WB saline by gently scraping the surface with a cell scraper, and the DNA was extracted before assay by real-time PCR.

In August and October 2011, 1 L water and sediment samples were collected from 10 lakes in Minnesota and 6 lakes in Florida. Each water and sediment sample was a composite from 4 sites along a beach area at each lake. Sediment samples were washed twice with 500 mL of WB saline and the supernatant was processed using the same procedure as the water samples (Figure 1). Each sample was concentrated by centrifugation as already described, then 1 mL of the resulting pellet was placed on an agar culture plate and the remaining pellet volume was processed using the IMS procedure. The resulting IMS concentrate was split in half: 50 μL was added to an agar culture plate and 50 μL was extracted and assayed using real-time qPCR.

Overall, not separated by ameba stage or water type, the IMS method was determined to recover 75 ± 17.7% of N. fowleri amebas (trophozoites and cysts separately) seeded in 5 mL water samples. As shown in Table 1, N. fowleri genotypes were more effectively recovered in trophozoite form than in cyst form. Average recoveries of N. fowleri were significantly higher for trophozoites (88 ± 8.4%) than cysts (61 ± 13.2%) when recovery data were analyzed without stratification for genotype or water type (P < 0.0001). No significant differences in recoveries were found between genotypes (P = 0.5741) or between the amended DI water and lake water (P = 0.4046).

The IMS method exhibited little binding to nonpathogenic Naegleria species and other free-living ameba. As shown in Table 2, out of the thirteen nontarget amebas tested, only Vahlkampfia lobospinosa, Naegleria lovaniensis, Naegleria italica, and Naegleria clarki showed a marginal degree (higher than 5% recovery) of cross-reactivity. The cross-reactivity efficiencies observed for non-N. fowleri amebas were significantly lower than these deserved for N. fowleri (P < 0.0001).

The qPCR assay detected all 4 genotypes of N. fowleri and when tested against the specificity panel of other Naegleria spp., and non-Naegleria amebas it did cross-react with one non-Naegleria ameba. The assay reported a false positive average CT values of 39.2 ± 1.0 for Hartmannella vermiformis (data not shown). The assay resulted in average CT values of 28.0 ± 3.1 for the 4 genotypes of N. fowleri (data not shown). Using triplicate tenfold dilutions of the DNA stock, a standard curve for the N. fowleri PCR TaqMan assay was developed and found to be linear over a range from 10⁰ to 10⁴ amebas per reaction (Figure 2). This testing indicated that the assay could be used to detect as low as 1 ameba per reaction. Using the formula, E = 10^(−1/slope) − 1, the calculated PCR efficiency for this assay was 96%.

The limit of detection for the sample processing method in conjunction with qPCR was determined, both for direct PCR without culture and PCR following culture. In these experiments, a concentration of 63 amebas/L could be consistently detected (3 of 4 samples positive) when IMS was used to process the samples and qPCR used for direct analysis (i.e., without prior culture) (Table 3). When IMS was not used, the direct PCR method detection limit appeared to be slightly higher. Similarly, IMS appeared to enable a lower method detection limit when culture of the pelleted samples was performed prior to PCR. Consistent PCR detection after culture was observed for a seed level of 388 amebas/L when IMS was used (4 of 4 samples positive) but was higher than 388 amebas/L when IMS was not used. The use of IMS also appeared to result in the N. fowleri cultures being capable of more easily replicating based on the substantially lower CT values associated with post-IMS cultures.

When the N. fowleri testing procedure was used to analyze 16 natural (nonseeded) lake water and 16 sediment samples, N. fowleri was detected by PCR in 6 sediment samples and 4 water samples (Table 4). Postculture detection appeared to be improved when IMS was used to process sediment samples (6 detections following IMS versus 2 detections when IMS was not used). However, the only two water samples that were positive for N. fowleri after culture were from the procedure when IMS was not used. Direct PCR was effective for rapidly detecting N. fowleri in only 3 of the 10 samples (water and sediment) that were found to be positive for N. fowleri but was the only technique that enabled detection of N. fowleri in two water samples (FL5 and FL6).

Using CT values from seeded and nonseeded sample testing, estimates of N. fowleri concentrations were made and compared to known concentrations when possible. Using this method, qPCR estimates were within an order of magnitude of the known amount of amebas in samples determined by microscopy (Table 5). Using the qPCR concentration estimates for the seeded samples, the sample processing method (including IMS) had an average recovery of 36 ± 16%. The 3 highest seed levels were also able to be directly counted using the hemocytometer and had an average recovery of 45 ± 5%. Figure 3 shows that the recovery efficiencies estimated using qPCR results were on average lower and more variable than the recoveries determined by direct microscopy counts.