Comparison of real-time PCR methods to detect Naegleria fowleri in environmental samples Open Access

Streby, Ashleigh (2012)

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Naegleria fowleri is a thermophilic free-living amoeba found in freshwater environments worldwide. It is the cause of a rare, but potentially fatal disease in humans known as primary amoebic meningoencephalitis (PAM). The purpose of this study was to compare four different real-time PCR methods (Jothikumar et al., Qvarnstrom et al., Puzon et al., Robinson et al.) for the detection of N. fowleri from surface water and sediment. It was hypothesized that either the Qvarnstrom et al. or Jothikumar et al. assay would perform the best based on the use of a specifically designed fluorescent reporter probe in these assays. The assays were compared in terms of thermodynamic stability, analytical sensitivity and specificity, detection limits, humic acid inhibition effects, performance with seeded environmental matrices and performance with samples previously tested as positive by the CDC Water Sanitation and Hygiene (WASH) Laboratory. Twenty-one amoeba isolates were included in the DNA panel used for analytical sensitivity and specificity analysis. N. fowleri genotypes I and III were used to determine detection limits whereas N. fowleri genotype I was used in humic acid inhibition and performance with seeded environmental matrix analyses. Two of the assays were removed from further investigation due to lower sensitivity (71%, Robinson et al.) and higher detection limit for N. fowleri genotype III (0.8 N. fowleri amoeba per real-time PCR reaction, Puzon et al.). Based on relatively equivalent outcomes at each stage of analysis, it was determined that both the Jothikumar et al. and Qvarnstrom et al. assays should be effective for use in future analyses with environmental matrices. The use of these assays should be useful contributors to studies investigating the potential risk factors for human exposure to N. fowleri.

Comparison of real-time PCR methods to detect Naegleria fowleri in environmental samples
Ohio State University
Thesis Committee Chair: Karen Levy, PhD
A thesis submitted to the Faculty of the
Rollins School of Public Health of Emory University
in partial fulfillment of the requirements for the degree of
Master of Public Health
in Global Environmental Health

Table of Contents

Table of Contents
1. Introduction & Background...1-7
2. Methods...8-14

a. Amoeba Isolates...8-9
b. Thermodynamic Stability of Assays...9
c. Real-Time PCR Assays...9-10
d. Assay Sensitivity and Specificity...10-11
e. Standard Curves and Limits of Detection...11
f. Humic Acid Inhibition Effects...12
g. Application of Real-Time PCR to Sediment and Water Samples...12-14
h. Real-Time PCR Analysis of Known Positive Samples...14
i. Statistical Analysis...14

3. Results...15-22

a. Thermodynamic Stability of Assays...15
b. Assay Sensitivity and Specificity...16-18
c. Standard Curves and Limits of Detection...18-20
d. Humic Acid Inhibition Effects...20-21
e. Application of Real-Time PCR to Sediment and Water Samples...21-22
f. Real-Time PCR Analysis of Known Positive Samples...22
g. Assay Choice...22-23

4. Discussion...24-28
5. Conclusions and Recommendations...28-29
6. References...30-32
7. Appendices...33-48

Table of Contents: Tables, Figures and Graphs
Figure 1: Experimental Plan for Comparison of N. fowleri Real-Time PCR Assays...7
Figure 2: Thermodynamic Stability of Assays...15
Figures 3A-3C: Standard Curves for N. fowleri Genotypes I and III...20
Figure 4: CT Value as a Function of Humic Acid Concentration...21
Table 1: Amoebae Isolates Used in Study...8
Table 2: Real-Time PCR Assays Included in Study...9
Table 3: Positions of Melt Curve Peaks for Robinson et al. Assay...17
Table 4: Sensitivity and Specificity of Real-Time PCR Assays...18
Table 5: Application to Environmental Sediment and Water Samples Results...21
Table 6: Assay Performance with N. fowleri Positive Samples...22
Table 7: Performance of Real-Time PCR Assays...23

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