Limits...
Improved Method for the Detection and Quantification of Naegleria fowleri in Water and Sediment Using Immunomagnetic Separation and Real-Time PCR.

Mull BJ, Narayanan J, Hill VR - J Parasitol Res (2013)

Bottom Line: Primary amebic meningoencephalitis (PAM) is a rare and typically fatal infection caused by the thermophilic free-living ameba, Naegleria fowleri.An immunomagnetic separation (IMS) procedure and real-time PCR TaqMan assay were developed to recover and quantify N. fowleri in water and sediment samples.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Foodborne, Waterborne and Environmental Diseases, Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases, 1600 Clifton Road, NE, Mail Stop D-66, Atlanta, GA 30329-4018, USA.

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.

No MeSH data available.


Related in: MedlinePlus

Recovery efficiency of N. fowleri in 1 L of seeded GA lake water.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3818898&req=5

fig3: Recovery efficiency of N. fowleri in 1 L of seeded GA lake water.

Mentions: 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.


Improved Method for the Detection and Quantification of Naegleria fowleri in Water and Sediment Using Immunomagnetic Separation and Real-Time PCR.

Mull BJ, Narayanan J, Hill VR - J Parasitol Res (2013)

Recovery efficiency of N. fowleri in 1 L of seeded GA lake water.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3818898&req=5

fig3: Recovery efficiency of N. fowleri in 1 L of seeded GA lake water.
Mentions: 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.

Bottom Line: Primary amebic meningoencephalitis (PAM) is a rare and typically fatal infection caused by the thermophilic free-living ameba, Naegleria fowleri.An immunomagnetic separation (IMS) procedure and real-time PCR TaqMan assay were developed to recover and quantify N. fowleri in water and sediment samples.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Foodborne, Waterborne and Environmental Diseases, Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases, 1600 Clifton Road, NE, Mail Stop D-66, Atlanta, GA 30329-4018, USA.

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.

No MeSH data available.


Related in: MedlinePlus