Naegleria fowleri : Highly Fatal, But Rare

Public Drinking Water System and

Wastewater System Operational Implications
A Project Funded by Arizona Water Institute
Al Brown, M.P.A., PI
Precious Biyela, Ph.D.
Hodon Ryu, Ph.D.
Absar Alum, Ph.D.
Morteza Abbaszadegan, Ph.D.
Bruce Rittmann, Ph.D.
Presented to the 2012 Gatekeeper Regulatory Roundup Conference
April 3, 2012, Scottsdale, Arizona
What is Naegleria fowleri?
• A free-living amoeba found in warm
freshwater (usually 77 – 90 °F)
• Not known to be in feces of mammals
• Causes Primary Amoebic
Meningoencephalitis (PAM)
• 97% case fatality rate
• Only 32 cases from 2001 – 2010 in U.S.
• Symptoms: 1 – 7 days after infection,
headache, fever, nausea, vomiting, stiff neck,
confusion, loss of balance, seizures,
hallucinations, death within 1 – 12 days
Physical characteristics

• Range of tolerance for trophozoites:

– Temperature: 53.6 – 131°F; optimum: 98.6 –
111°F
– pH: 4.0 – 9.1
– Size: 10 – 30 µm; cysts: 7 – 14 µm
– Found in aquatic sediments, water, biofilm, soil
– Not especially resistant to desiccation
Risk Factors
• Route of infection is through the nose
– Forceful entry of water by jumping, diving, water
skiing etc. is often reported
• Warm freshwater lakes, rivers, ponds, hot
springs
• Unchlorinated or poorly maintained
swimming pools
• Jacuzzis, hot tubs, spray fountains or water
play areas
• Neti Pots or similar nasal/sinus rinses
• Hot water tanks less than 116 ° F
Neti Pots
Risk Factors Continued

• Naegleria fowleri can in principle be used

as a biological agent against humans
• Requires biosafety level 2 precautions for
researchers
• Drinking water and wastewater system
operators should maintain proper personal
hygiene practices
Arizona Drinking Water System Cases
• On October 12 and 13, 2002, two five-year
old boys died from presumed bacterial
meningitis within 4 hours of each other at two
Phoenix area hospitals
• Epidemiological investigation suggested
PAM; common source of exposure: Rose
Valley Water Co. unchlorinated water
• On October 18, 2002 water samples from
RVW Co. were + for total coliform bacteria
• On October 21, 2002, both cases were
confirmed as PAM
Water System Investigation

• On November 1, 2002, RVW Co. reported +

result for a Naegleria fowleri test
• On November 2, 2002, boil water order
issued and emergency connection to City of
Peoria Water system was initiated
• On November 3 – 4, 2002, CDC and
Maricopa County collect 100 gallon samples
from RVW Co. water system
• On or about December 1, 2002, CDC
confirms Naegleria fowleri in RVW Co. water
 2002 Investigation Results

Sample location Presence of N. fowleri

• RVW Tank 1 • Negative
• RVW Tank 2 • Negative
• RVW Tank 3 • Positive
• RVW Well 2 • Negative
• RVW Well 3 • Negative
• Peoria Well • Positive
• Refrigerator filter • Positive
Rose Valley Water System, Peoria, AZ, ca. 2002
RVW Tank #2

RVW Tank #1
RVW Tank #3

Small tank
not in use
since June
2002

RVW Well #2

POE 002
Rose Valley Water System, Peoria, AZ, Tank No. 1; ca. 2002
Rose Valley Water System, Peoria, AZ, Tank No. 3; ca. 2002

Tank Wall #3

Ladder

Float for water level gage

Floor on Tank #3
 Close up on Outlet for Tank #3

Flow of Water leaving the tank

Tank #3 Floor
Temperature Distribution of Groundwater Samples; 2002

200
175
Number 150
of wells 125
100
75
50
25
0
>6

60

70

80

90

10

<1
0

0t

10
to

to

to

to

o
70

80

90

10

11
0

0
Temperature
Project Hypotheses

• Biofilms in drinking water systems provide

a substrate for growth and survival of
Naegleria fowleri
• Factors that reduce the accumulation,
survival and spread of N. fowleri include:
– Chemical and biological factors
– Plumbing construction materials
– Operational practices
Research approaches

• Test for presence of N. fowleri in samples

from drinking water systems before and
after an operational practice
• Test for survival and growth of N. fowleri on
lab scale pipe loop biofilm accumulation
• Evaluation of selected chemical and
biological parameters as indicators of N.
fowleri
Water System Selection

• City of Peoria volunteered to serve as the

chlorinated groundwater source (pop.
145,000)
• U of A Maricopa Agricultural Center (MAC)
volunteered to serve as the unchlorinated
groundwater source (pop. 100)
AWI Project; Field Sample Matrix: Locations

• Distribution points
• Water storage tanks
• Valves
• Hydrants
• Meters
• Dead ends
Field Samples

• 8 - 40 L water samples
• 20 – 500 ml biofilm samples
• 3 – 1 L water samples per location
• Samples were collected before and after
an operational practice employed by the
water system operators
Field Sample Test Parameters

• pH
• Temperature
• DO
• Total Chlorine
• Free Chlorine
MAC Valve Section Sample Location
MAC Valve Section Biofilm Sample
MAC Hydrant Flush Operational Practice (OP)
Peoria Hydrant: First Flush
Peoria Hydrant Biofilm Sample Collection
Peoria Hydrant Biofilm
Peoria Hydrant Biofilm
City of Peoria Water Reservoir
Biofilm Sample from Water Reservoir, Before Operational Practice
Biofilm/Sludge from Water Reservoir Bottom, Before OP
Biofilm/Sludge Sample from Water Reservoir Bottom, Before OP
Field Sampling Results
„ A total of 28 (8 40-L water and 20 biofilm) samples
were collected and processed.
„ All samples were analyzed for amoebic activities using a
viability assay.
„ Amoebic activity was observed in two of 8 water samples
and 14 of 20 biofilm samples.
„ 31% of the sampling locations that originally had amoebic
activity showed no such activity after the best operational
practices such as flushing
„ For confirmation, DNA was extracted from the amoebae -
positive samples and stored at -80 C for a molecular
analysis.
„ No field samples were + for Naegleria fowleri
Field Sample Results

• 67% of both Peoria and MAC biofilm

samples were + for amoebic activity
• Legionella was present in 64% of biofilm
samples with amoebic activity
• Acanthamoeba was not found
 Pilot-scale Simulator: Cast Iron & PVC
ƒ N. fowleri spiked : 3.5x107
1TCID
50
ƒ Estimated number of N. fowleri in
flowing water: 103 TCID50/mL
ƒ Weekly water sampling from both
pipes until no amoebic activity is
observed (natural die-off, >4.5
log10)

Biofilms from both pipes were

assayed for amoebic activity
at the end of simulator run
1 Tissue Culture Infective Dose per ml
Slide provided by Morteza Abbaszadegan, Ph.D.,
Director, NSF Water Quality Center
 Cast Iron
7 years old 5 months old

5-month old biofilm samples from newly installed pipe segments were collected
and analyzed for amoebic activity.

Slide provided by Morteza Abbaszadegan, Ph.D.,

Director, NSF Water Quality Center
 PVC

7 years old

Slide provided by Morteza Abbaszadegan,

Ph.D., Director, NSF Water Quality Center
 Amoeba
Cyst

Trophozoites

Slide by Hodon Ryu, Ph.D.

Summary of Simulator Results
„ Amoebic activity was observed in both water
and biofilm samples after 5-month simulator
run.
„ N. fowleri was present in all 20 water
samples
„ N. fowleri was observed in biofilm after 5
months
„ This supports the hypothesis that biofilm can
sustain N. fowleri
Chemical and Biological Parameters

• Temperature • Total nitrogen

• pH • Total ammonia
• Dissolved oxygen • Ammonium
• Total chlorine • Nitrite
• Free chlorine • Nitrate
• Dissolved organic • Heterotrophic plate
carbon (DOC) count
• Biodegradable
dissolved organic
carbon (BDOC)
Chemical and Biological Field Sample Results
• DOC
– Lower in unchlorinated system
– Lower with more residence time/stagnation
– Operational practices had insignificant effect
• BDOC
– Lower in unchlorinated system
• HPC
– Higher in unchlorinated system
– Higher with residence time/stagnation
– Lower after flushing
• Cl residual
– Lower with residence time/stagnation
• NO3
– Lower in unchlorinated system
– Lower after flushing in both systems
Chemical and Biological Pipe Loop Results

PVC VS. Cast Iron

• DOC lower over time • DOC lower over time
• BDOC lower over time • BDOC lower over time
• HPC higher • HPC lower
• NO3 lower • NO3 higher
• Biofilm thickness • Biofilm thickness
less/even greater/uneven
• Biofilm bacteria • Biofilm bacteria
higher/unit area lower/unit area
 Significant Findings
• N. fowleri colonized biofilms in the laboratory
setting
• Amoebic activity was present in 67% of water
distribution biofilm samples
• Chlorine residual did not eliminate HPC
• Amoebic activity increases with:
– Lack of chlorine
– Stagnation/lack of flushing
– Low DOC
– Low BDOC
– Higher HPC
Significant Findings

• A positive relationship exists between

water quality deterioration and the
presence of amoebic activity
• The unidirectional flushing operational
practice had only a modest impact on
improving water quality indicators
Recommendations for drinking water systems

• Work towards elimination of the main

causes of bacteriological growth in
finished water. The primary sources of
bacteria/biofilm growth are BDOC and
ammonia.
• Additional studies on amoebic activity are
needed through samples of several
different water systems.
Wastewater Implications

• Naegleria fowleri is not likely to be found in

septic tank sludge or effluent because it is not
amplified by mammals or known to be
transmissible via the fecal/oral pathway
• Septic tank sludge and effluent provide a
suitable source of nutrients for Naegleria
fowleri to use for growth in soil and water
– High BOD (> 50 mg/L in effluent)
– High total nitrogen (> 35 mg/L in effluent)
– High coliform (650,000 CFU/100 ml in effluent)
Septic tank effluent transport

• Coliform bacteria were found to move up to

2723 ft in sand and gravel
• Coliform bacteria were effectively filtered out
(within 100 feet) by fine particle soil horizons
such as fine sandy loam and fine sand
• Virus removal efficiency is unknown
Causes of Waterborne Disease Outbreaks at Groundwater Sources

• Seepage of sewage
• Chemical contamination
• Surface water runoff
• Interruption of disinfection
• Inadequate disinfection
• Poor system O & M
Recommendations for wastewater systems

• Proper design, construction, operation and

maintenance of wastewater systems will
minimize the risk of exposure to infective
doses of Naegleria fowleri
• Minimize time of exposure of onsite
wastewater system effluent surfacing
through rapid enforcement by regulatory
agencies or rapid voluntary remedial
response actions by responsible parties
Protozoan parasites

• Giardia lamblia
• Entamoba histolytica
• Balantidium coli
• Cryptosporidium parvum
• Cyclosporidium
• Microsporidia
• Naegleria fowleri
Cryptosporidium parvum

• Resistant to chlorine
• Infective dose as low as 1 organism
• Vegetables and unpasteurized fruit juices
are the most commonly reported food
associations
• Water is the primary source of infection
• No effective treatment exists

FDA, 2007, Bad Bug Book, http://www.cfsan.fda.gov/~mow/chap24.html

CDC, 2008, http://www.cdc.gov/crypto/bwa/commercial.html#cook
Excysting Sporozoites of C. parvum

http://www.stanford.edu/class/humbio103/ParaSites2001/crypto/cryptoscape.jpg
Protozoan Research Findings
ƒ Cryptosporidium oocysts and Giardia cysts were
detected in 16% (12/77) and 43% (33/77) of reclaimed
water samples.

ƒ Combined risks of infection from both parasites

– Utilities using only chlorination: frequent failures to meet
the annual acceptable risk of 1.00E-04 (1:10,000) for
drinking water
– Utilities using a combination of UV disinfection and
chlorination: <1.00E-04

Source: Morteza Abbaszadegan, Hodon Ryu, and Absar Alum, ASU NSF Water
Quality Center, 2006
 References
Abbaszadegan, M, Ryu, H., and Alum, A., 2006, ASU NSF Water
Quality Center, Microbial characterization and the risk of infection by Cryptosporidium
and Giardia in non-potable reclaimed water

Begley, Sharon. (2011) Brain-eating amoeba cases puzzle and worry scientists, The
Daily Beast, December 23, 2011,
http://www.thedailybeast.com/articles/2011/12/23/brain-eating-amoeba-cases-puzzle-
and-worry-scientists.html

Bitton, G. 2005, Wastewater Microbiology, Third Edition, Hoboken, N.J., Wiley & Sons

Bitton, G., 1978, Survival of Enteric Viruses, in Mitchell, R., 1978, Water Pollution
Microbiology, V2, New York, Wiley & Sons

Biyela, Precious, T., Ryu, H., Brown, A., Alum, A., Abbaszadegan, M., and Rittmann,
B. (2012). Distribution systems as reservoirs of Naegleria fowleri and other amoebae.
Journal – American Water Works Association, doi:
http://dx.doi.org/10.5942/jawwa.2012.104.0007
 References
Biyela, Precious, T., Ryu, H., Brown, A., Alum, A., Abbaszadegan, M., and Rittmann,
B. (2012). Evaluating proposed operational practices for control of Naegleria fowleri
in Arizona’s public drinking water systems. (2011). Arizona Water Institute, Project
Final Report No. AWI 08-04, Arizona Department of Environmental Quality.

Chihhao, Fan, Chang, F.C., Ko, C.H., Teng, C.J., Chang, T.C., Sheu, Y.S. (2009).
Treatment of septic tank effluents by a full-scale capillary seepage soil biofiltration
system, Journal of Environmental Health, Vol. 71, No. 7, PP 56 – 60.

De Michele, E. 1974, Water Reuse, Virus Removal and Public Health, in Malina, J. F.
and Sagik, B.P. Virus Survival in Water and Wastewater Systems, 1974, Center for
Research in Water Resources, University of Texas at Austin

Friis, R.H., 2007, Essentials of Environmental Health, Sudbury: Joes and Bartlett

Hammer, Mark, J., Hammer Jr. M. (2004). Water and Wastewater Technology, Fifth
Edition, Pearson Prentice Hall, Columbus, Ohio

Hagedorn, C. 1984, Microbiological Aspects of Groundwater Pollution due to Septic

Tanks, in Bitton, G. and Gerba, C.P., Groundwater Pollution Microbiology, New York,
Wiley & Sons
 References
Heymann, David, L. (2008). Control of Communicable Diseases Manual, 19th
Edition. American Public Health Association, Washington D.C.

Salvato, Joseph, et. al., Environmental Engineering and Sanitation, 5th edition,
John Wiley & Sons, 2003

Sifuentes, Laura, Gerba, C., and Rock, C. (2011) Playing safe in natural waters:
how to protect yourself from Naegleria fowleri when you go swimming. The
University of Arizona, Publication AZ1545,
http://cals.arizona.edu/pubs/water/az1545.pdf

U.S. Centers for Disease Control (CDC), 2012, Naegleria faqs,

http://www.cdc.gov/parasites/naegleria/faqs.html

U.S. Centers for Disease Control (CDC), 2009, http://www.cdc.gov/foodsafety/

U.S. CDC, 2008, http://www.cdc.gov/crypto/bwa/commercial.html#cook

U.S. CDC/MMWR, 2006,

http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5723a2.htm
 References
U.S. Food and Drug Administration (FDA), Bad Bug Book,
http://www.cfsan.fda.gov/~mow/chap15.html

U.S. FDA, 2007, Food Protection Plan,

http://www.fda.gov/oc/initiatives/advance/food/plan.html

Weisbuch, Jonathan, B. (2003) Naegleria fowleri in Arizona: Tracking a Deadly

Ameba, (not published)

Wright, Richard, and Boorse, D., (2011), Environmental Science: Toward a

Sustainable Future, Eleventh Edition, Pearson Prentice Hall, Upper Saddle River,
New Jersey
QUESTIONS AND DISCUSSION

al.brown@asu.edu
Briefbiography

Mr.AlBrown,M.P.A.,servesasDirectorofEnvironmentalResearchInitiativesandLecturer.Mr.Brown
isaformerDirectoroftheMaricopaCountyEnvironmentalServicesDepartmentwherehedirected
multipleenvironmentalprograms.HehasservedasamemberofADEQ’sNaegleriaAdvisoryPanel.Mr.
Brownhasover35yearsofenvironmentalandemergencymanagementexperienceincludingfood
safety,waterquality,airquality,hazardouswaste,solidwasteandwastewatermanagement,vector
control,environmentalremediationanddirectionofenvironmentalemergencyresponses.Whileat
ASU,Mr.Brownhastaughtupperdivisionundergraduateandgraduatelevelcoursesinenvironmental
health,waterandwastewatertreatment,environmentalmanagement,environmentalleadership,
sustainabilityandenvironmentalregulations.HewasthePrincipalInvestigatorforaresearchproject
fundedbytheArizonaWaterInstitutethatevaluatedtheeffectivenessofpublicdrinkingwatersystem
operationalpracticesforthecontrolofN.fowleri.Mr.BrowniscurrentlythePrincipalInvestigatorfora
FEMAprojecttodevelopfournewonlinecoursesforcampusemergencycommunicatorsand
managers.Mr.BrownisalsoaCoPIforaprojecttoimproveenergyassuranceandenergyemergency
planningfortheStateofArizona;andaCoPIforaprojecttotestanewarsenicremovaltechnologyon
smalldrinkingwatersystems.Mr.BrownhasservedASUemergencymanagementfunctionsbyhelping
developtheuniversity’spandemicinfluenzaplan.Inaddition,Mr.Brownconductsnondegreetraining
forASUintheareasofairpollutioncontrolandOSHAHazWOpERclasses.