Wisconsin WAHTER Study

Wisconsin Water And 

Health Trial for Enteric 

Risks (WAHTER Study)

Part 1:

Risk of Illness from Municipal 

Groundwater Consumption

Mark Borchardt, PhD
Wisconsin WAHTER Study

National Estimate of Waterborne 

Disease in the United States

“The Safe Drinking Water Act (SDWA) 

Amendments of 1996, section 1458(d), provides 

that within two years of enactment the 

Environmental Protection Agency (EPA) and the 

Centers for Disease Control and Prevention 

(CDC) will conduct pilot waterborne disease 

occurrence studies for at least five major U.S. 

communities or public water systems.”

Federal Register/Vol 63, August 11, 1998
Wisconsin WAHTER Study

What Fraction of Acute Gastrointestinal 

Illness in the USA is Attributable to 

Drinking Water?

• 12% - median attributable risk 

percent of five published 

household intervention studies 

(Colford et al. 2006)

• 8.5% - Monte Carlo simulation 

based on Canadian household 

intervention studies (Messner et 

al. 2006)

• Important data gaps: 

Groundwater risks and 

Distribution system risks
Wisconsin WAHTER Study

Waterborne Disease Outbreaks 

in the USA

• From 1971 to 2004 there were 738 outbreaks 

associated with an infectious agent in drinking water; 

60% of the outbreaks were attributable to groundwater 

• Pathogen in about 50% of outbreaks is unknown and 

assumed viral

• Of all outbreaks reported, the number due to 

distribution system failures is increasing: 

12% during 1981 - 1990; 21% during 1991 - 2000; 

39% during 2001 - 2004

Summarized from CDC reports, e.g., MMWR, 2006, 55(SS12);31-58
Wisconsin WAHTER Study

Virus Occurrence in Groundwater 

in the USA

 Tested 448 municipal wells in 35 states, 31.5% positive 

for viruses (Abbaszadegan et al 1998)

 Tested 30 municipal wells in 17 states, 23% positive for 

enteroviruses (Lieberman et al 1999)

 Tested 29 wells and 72% were virus-positive (Fout et al 

2003)

 Viruses in Wisconsin groundwater: Private domestic 

wells (8%), City of La Crosse wells (83%), and City of 

Madison wells (66%) were virus-positive (Borchardt et al 

2003, 2004, 2007)

 Occurrence and Monitoring Document for the Final 

Ground Water Rule: at some point in time 27% of public 

water supply wells are virus-positive (EPA 2006)
Wisconsin WAHTER Study

Virus Sources and Infiltration 

Routes into Groundwater
Wisconsin WAHTER Study

Virus Subsurface Transport 

Potential

• Small size (∼ 50 nm) and negative charge favor 

movement through soil 

• Survivability favored by low temp, moisture, and 

absence of UV light

• Documented transport: penetration as deep as 72 m, 

horizontal migration up to 1600 m
Wisconsin WAHTER Study

Enteric Viruses –

Clinical Significance

• Enteroviruses: fever, “summer cold”, diarrhea, hand, 

foot, mouth disease, conjunctivitis, meningitis, 

myocarditis, poliomyelitis, diabetes? chronic fatigue 

syndrome?

• Rotavirus: severe diarrhea and vomiting, 50,000 

hospitalizations/year in US

• Hepatitis A virus: gastroenteritis, hepatitis, fatality rate of 

2.7% in people > 49 years of age

• Noroviruses: gastroenteritis, “the flu”

• Adenoviruses: diarrhea, acute respiratory illness, 

pneumonia, conjunctivitis, neurological diseases, obesity?
Wisconsin WAHTER Study

Health Risk or Non-Issue?

• So viruses are present in public water 

supply and domestic wells ...

• Does it matter?

• Is there any effect on public health?
Wisconsin WAHTER Study

Wisconsin WAHTER Study

Funded by the US EPA STAR Program

Study Period: 2005 – 2008

Collaborators include UC-Davis 

and UW-Madison
Wisconsin WAHTER Study

Study Objectives

1. Determine the association between tap water virus 

concentrations and community illness rates

2. Estimate the risk of acute gastrointestinal illness (AGI) 

from drinking municipal water from groundwater 

sources

3. Accounting for any risk contributed by groundwater, 

estimate the AGI risk contributed solely by 

contaminated distribution systems

4. Determine the association between viruses in 

distribution systems and utility operation and 

maintenance procedures
Wisconsin WAHTER Study Design

Intervention trial in 14 groundwater-source communities

Communities Set 2

Communities Set 1 Communities Set 2

Communities Set 1

UV Light

Intervention

Control

Two 3-month 

Surveillance

Periods

Cross-over

Period

Two 3-month 

Surveillance

Periods
Wisconsin WAHTER Study

WAHTER Study Participating 

Communities

Populations: 1,200 – 8,300

Number Wells: 2 – 5

Pumpage: 0.13 – 2.1 MGD

Hydrogeology: sand, 

sandstone, limestone

No surface water influence

No disinfection
UV Intervention Effect

Well

Distribution System Pipes

Distribution System Pipes

UV 

Disinfection

No Intervention

Groundwater

Groundwater

Well

Fecal Contamination

Fecal Contamination

NonIntervention 

Communities 

illness due to 

groundwater 

plus 

distribution 

system

Intervention 

Communities 

illness due to 

distribution 

system only

Intrusions

Intrusions
UV Disinfection

performance through technology

W59 N249 Cardinal Ave.

Cedarburg, Wisconsin

262-377-6360

262-377-1515

www.energenecs.com

Service 24/7

from

Regional Sites

WEDECO/ITT 

Corporation

Staab Construction

Equipment

Installation

Design Service
UV dose = 50 mJ/cm2



Wisconsin WAHTER Study

Epidemiological Study Design

• Eligibility: family served by study community’s water 

system and have at least one child 6 months to 12 

years old

• Exclusions: chronic GI illness; child attends

daycare or school outside of community more than 

20 hrs/week

• Participants submitted an illness symptom checklist 

every week
Wisconsin WAHTER Study

Epidemiological Study Design

(cont)

• Acute gastrointestinal illness (AGI) surveillance for four 

12 week periods, spring and autumn 2006 and 2007

• AGI defined as ≥ two episodes loose watery stools OR ≥ 

one episode vomiting in 24 hour period

• Episodes separated by ≥ 6 symptom-free days

• Person-time estimated from nights slept away from 

home, self-reported on symptom checklist

• Outcome measure: Number AGI episodes/person-year 

for each community and surveillance period
Wisconsin WAHTER Study

Participating Households’ 

Characteristics

• Beginning enrollment: 

621 households

• Ending enrollment: 

440 households
Wisconsin WAHTER Study

Study Subjects’ Characteristics

• Beginning enrollment: 

1,079 children, 580 

adults

• Ending enrollment: 765 

children, 413 adults
Wisconsin WAHTER Study

Water Monthly Sampling Plan

• Time-composite, 

continuous 30 day 

samples from two points 

in distribution system

• All operating wells and before and after UV disinfection 

units

• Distribution system samples (up to 8 per community) 

collected from household taps
Wisconsin WAHTER Study

Virus Captured by Glass Wool 

Filtration

Volume sampled (liters)

Mean = 860

Range: 76 – 2,067

N = 1,786
Wisconsin WAHTER Study

Virus Laboratory Analyses

• Water samples analyzed for six virus groups: 

enteroviruses, adenoviruses, GI and GII noroviruses, 

hepatitis A virus, and rotavirus

• Viruses quantified by qRT-PCR and qPCR using 

LightCycler 480 system and TaqMan probes

• Extensive QA/QC procedures and PCR inhibition 

quantified and corrected for every sample

• Adenovirus and enterovirus infectivity evaluated by 

cell culture

• Adenovirus and enterovirus serotype determined by 

sequencing
Wisconsin WAHTER Study

Determine the association between tap 

water virus concentrations and community 

illness rates

Objective 1
Wisconsin WAHTER Study

Virus Types, Frequencies, and 

Concentrations in Distribution Systems

N = 1,204 samples
Wisconsin WAHTER Study

Relating Viruses to AGI Incidence

Statistical Approach

Poisson regression to relate AGI to viruses by community 

and surveillance period (14 communities by 4 periods = 56 

data points)

Two set of models: 1) Unadjusted; 2) Adjusted with random 

effects for community and 3-month reporting period

All models include multiplicative overdispersion parameter

Three virus measures: arithmetic mean concentration, 

proportion positive samples, maximum concentration

Virus measures initially represented by splines, which 

indicated linear representations were adequate

Rate ratios represented by splines to visualize the virus 

concentration at which the AGI rate is significantly elevated
Wisconsin WAHTER Study

Total Virus Concentration in 

Tap Water and AGI Incidence

All ages 

Unadjusted

Virus conc for significant rate ratio = 2.1

P-value = 0.01

Significant virus conc = 2.2
Wisconsin WAHTER Study

All ages 

Adjusted for 

community and 

period

Total Virus Concentration in 

Tap Waterand AGI Incidence

Virus conc for significant rate ratio = 2.2

P-value = 0.10
P-Values Indicating Strength of Association 

between Community AGI Incidence and Three 

Measures of Virus Contamination: Mean 

Concentration, Proportion Samples VirusPositive, and Maximum Concentration

P-values < 0.1

< 0.05

< 0.01

< 0.001
Wisconsin WAHTER Study

All ages 

Unadjusted

G1 Norovirus Concentration in 

Tap Waterand AGI Incidence

Virus conc for significant rate ratio = 0.001

P-value = 0.0005
Wisconsin WAHTER Study

0

20

40

60

80

100

120

Spring ’06 Fall ’06 Spring ’07 Fall ’07

Study Period

Adenovirus

Enterovirus

GI Norovirus

Hepatitis A

Number of Positive Samples

Virus Types Detected in the 

Distribution Systems
Wisconsin WAHTER Study

All ages 

Adjusted for 

community and 

period

For comparison, 

the rate ratio is for 

all viruses, all 

ages, adjusted for 

community and 

period

Adenovirus Concentration in 

Tap Water and AGI Incidence

P-value = 0.10
Adenovirus Serotypes by 

Surveillance Period in Tap Water

0

20

40

60

80

100

120

1 2 3 4

Adenovirus 41

Adenovirus 40

Adenovirus 6

Adenovirus 5

Adenovirus 2

Unable to sequence

Period 1 Period 2 Period 3 Period 4

Number of Samples

Wisconsin WAHTER Study
Wisconsin WAHTER Study

Enterovirus Concentration in 

Tap Water and AGI Incidence

Adults

Adjusted for 

community and 

period

P-value = 0.003

Virus conc for significant rate ratio = 0.6
Wisconsin WAHTER Study

0

10

20

30

40

50

60

1 2 3 4

Echovirus 30

Echovirus 18

Echovirus 13

Echovirus 11

Echovirus 9

Echovirus 6

Echovirus 3

Coxsackie B5

Coxsackie B4

Coxsackie B3

Coxsackie B2

Coxsackie A9

Coxsackie A16

Unable to sequence

Number of Samples

Period 1 Period 2 Period 3 Period 4

Enterovirus Serotypes by 

Surveillance Period in Tap Water
Infectious Enteroviruses by 

Integrated Cell Culture-PCR 

120

100

80

60

40

20

0

120

100

80

60

40

20

0

Wisconsin WAHTER Study

0

20

40

60

80

100

120

Wells1 Post 2 3 -UV

No. of Samples ICC-PCR Positive 

No. of Samples qPCR Positive 

Distribution 

System
Wisconsin WAHTER Study

Objective 1 Summary

• Virus levels in tap water were significantly associated with 

community rates of AGI; the higher the virus concentration, 

the more illnesses in the community

• GI Norovirus measures (mean, proportion positive, and 

maximum) were all strongly associated with AGI

• Adenovirus detects were common, but concentrations were 

low and unassociated or inversely associated with AGI

• Enteroviruses were associated with adult AGI in certain 

periods, when echoviruses were predominant

• The risk of AGI was significantly elevated when the 3-month 

average virus concentration was approximately 2 viruses/liter

• qPCR detection of viruses in non-chlorinated drinking water 

is meaningful and has value for understanding waterborne 

AGI risk
Wisconsin WAHTER Study

Estimate the risk of acute gastrointestinal 

illness (AGI) from drinking municipal water 

from groundwater sources

Objective 2

DS

DS WW

Other Other

Incidence 

attributable to 

well water

Risk during 

NOUV

Risk during 

UV
Wisconsin WAHTER Study

Viruses in the Study Wells

In the 14 study communities, of all 36 wells tested, 34 were 

virus-positive (139 positive samples out of 392 (36%))
Estimating Attributable Risk of 

Groundwater-borne AGI:

Unadjusted Analysis

Communities Set 2

Communities Set 1 Communities Set 2

Communities Set 1

UV Light

Intervention

Control

AGI

Surveillance

Cross-over

Period

AGI 

Surveillance

• Attributable risk estimated as 

where IiC is the illness incidence in the ith community during the control period 

and IiI is the illness incidence in the ith community during the intervention 

period.

• H0 = the true difference in AGI incidence between intervention and control 

periods is 0.

• Test H0 using the permutation test

( )



 





∑ − =

14

14 1

1

i

iC iI I I
Estimating Attributable Risk of 

Groundwater-borne AGI:

Adjusting for Covariates

• Fit a Poisson regression model of AGI as related to covariates: 

age, gender, day care attendance, year, season, and well virus 

concentrations.

• Derive from the model the expected AGI counts for each record 

in the source data set. 

• Aggregate expected and observed counts to the level of 

community and intervention status (UV or control).

• Compute a residual at the level of community and intervention 

status [(observed – expected)/person time]

• Input the residuals into the permutation test; [(residual in the 

control period) – (residual in the intervention period)] is computed 

for each community

• The intervention effect (i.e., attributable risk) is the average of 

these differences across communities 
Intervention Effect

(i.e., Attributable Risk) for 

Groundwater-borne AGI

All analyses weighted by sample size (i.e., person-time)

*Adjusted for age, gender, day care attendance, year, season, and 

virus concentrations in the communities’ wells
Wisconsin WAHTER Study

Attributable Risk Percent for 

Groundwater-borne AGI in Adults

Weighted mean AGI rate in adults in 

control communities = 2.47

AR% = (0.22/2.47) x 100 = 9%

95% Confidence Interval: - 5% - 22%
Wisconsin WAHTER Study

Does Groundwater-borne Illness 

Risk Meet EPA Standards?

• Acceptable EPA risk for waterborne disease is 

1 infection in 10,000 people/year

• Assume every infection leads to an illness, then the 

acceptable illness rate is 0.0001 illness/person-year

• WAHTER Study measured in adults 0.22 illness/personyear attributed to groundwater

• 2,200 times higher than EPA acceptable risk
Wisconsin WAHTER Study

Intervention Effect Only 

Observed in Adults, Why?

• Groundwater-borne transmission was 

minimal to zero

•Statistical power insufficient

• Drinking water consumption differed 

between adults and children

• Illness susceptibility to viruses in 

groundwater differed between adults and 

children

• Viruses contaminated drinking water in the 

distribution system downstream of the UV 

intervention
Wisconsin WAHTER Study

Power Analysis for Overall Comparison 

of Intervention Versus Control
Wisconsin WAHTER Study

Intervention Effect Only 

Observed in Adults, Why?

• Groundwater-borne transmission was 

minimal to zero

•Statistical power insufficient

• Drinking water consumption differed 

between adults and children

• Illness susceptibility to viruses in 

groundwater differed between adults and 

children

• Viruses contaminated drinking water in the 

distribution system downstream of the UV 

intervention
Wisconsin WAHTER Study

Tap Water Norovirus Concentration 

and AGI Incidence in Children

Insert Burney’s figures

P-value = 0.008

Virus conc for significant rate ratio = 0.8
Wisconsin WAHTER Study

Intervention Effect Only 

Observed in Adults, Why?

• Groundwater-borne transmission was 

minimal to zero

•Statistical power insufficient

• Drinking water consumption differed 

between adults and children

• Illness susceptibility to viruses in 

groundwater differed between adults and 

children

• Viruses contaminated drinking water in the 

distribution system downstream of the UV 

intervention
P-Values Indicating Strength of Association 

between Community AGI Incidence and Three 

Measures of Virus Contamination: Mean 

Concentration, Proportion Samples VirusPositive, and Maximum Concentration

P-values < 0.1

< 0.05

< 0.01

< 0.001
Period 1 Only

Attributable Risk for Groundwaterborne AGI

All analyses weighted by sample size (i.e., person-time)

*Adjusted for age, gender, day care attendance, and virus 

concentrations in the communities’ wells
Wisconsin WAHTER Study

Intervention Effect Only 

Observed in Adults, Why?

• Groundwater-borne transmission was 

minimal to zero

•Statistical power insufficient

• Drinking water consumption differed 

between adults and children

• Illness susceptibility to viruses in 

groundwater differed between adults and 

children

• Viruses contaminated drinking water in the 

distribution system downstream of the UV 

intervention
Wisconsin WAHTER Study

Total Virus Concentration and AGI 

Incidence by UV Treatment Status
Wisconsin WAHTER Study

Objective 2 Summary

• The overall UV intervention effect in reducing AGI was 

strongest in adults; observable (albeit non-significant) AGI 

reductions occurred in the other age groups only in Period 

1 when GI norovirus was abundant

• AGI attributable risk from groundwater for adults was 0.22 

episodes/person-year, meaning 9% of all AGI in adults 

during the study period was from contaminated 

groundwater

• The AGI fraction attributed to groundwater likely depends 

on population characteristics (e.g., age group, immune 

status) and temporal factors such as year, season, viruses 

circulating in the population, and weather.
Wisconsin WAHTER Study

Objective 4

Determine the association between viruses 

in distribution systems and utility 

operation and maintenance procedures
Wisconsin WAHTER Study

Experimental Rationale

• Because the study communities do not have a chlorine residual, 

to the extent UV disinfection is effective, all viruses detected at 

household taps must have entered through the distribution 

system

• Written surveys on events related to distribution system 

operation and maintenance completed regularly by water 

operators

Well

Intrusions

Community-Wide Intervention with UV Disinfection

Distribution System Pipes

UV Disinfection

Groundwater Fecal Contamination
Total Viruses Genomic Copies/L

Sample 

Location

Number 

Samples Mean Median

75th

Percentile

95th

Percentile Maximum

Wells Pre UV 192 6.2 0 0.4 16.6 264.4

Wells Post UV 191 0.03 0 0 0.2 1.8

Distribution 

System

585 0.3 0 0 1.1 26.3

Virus Concentrations –

Summary Statistics for Year 1
Wisconsin WAHTER Study

Virus Concentrations from 

Source to Tap
Event

Proportion 

Samples

Virus Positive

P-Value 

Proportions 

Comparison

Mean Virus

Conc (GC/L)

Among Positives

P-Value

Means 

Comparison

Times 

Chlorinated

>0

0

0.19

0.09

0.086 3.7

2.1

0.284

Water Main 

Breaks

>0

0

0.17

0.12

0.412 3.4

2.9

0.746

Water Main Cut 

Open

>0

0

0.21

0.09

0.038* 4.4

1.1

0.028*

Water Tower 

Maintenance

>0

0

0.19

0.11

0.23 2.9

3.3

0.428

Cross 

Connections

>0

0

0.19

0.13

0.647 2.7

3.1

0.835

Times Hydrant 

Flushed

>0

0

0.15

0.11

0.575 3.1

3.1

0.876

Times Valves 

Exercised

>0

0

0.09

0.15

0.370 1.2

3.5

0.096

Total Coliform 

Detects

>0

0

0.21

0.10

0.126 4.3

2.0

0.159

Association of Events & Viruses in 

Distribution Systems
Wisconsin WAHTER Study

Thoughts, Opinions, Gut Feelings

• There is a significant public health risk from drinking nondisinfected groundwater. These study communities should be 

disinfecting.

• qRT-PCR measured viruses in non-chlorinated tap water are 

significantly associated with community illness rates. I will not 

drink water that is virus-positive by qRT-PCR.

• Viruses in groundwater and distribution systems are highly 

variable in space and time. Frequent virus sampling is necessary 

to reveal patterns.

• Viruses enter distribution systems and in some system locations 

present considerable risk.
Wisconsin WAHTER Study Team
Wisconsin WAHTER Study

Acknowledgments

Laboratory

Susan Spencer

Phil Bertz

Matt Volenec

Epidemiology

Ed Belongia, M.D.

Sandy Strey

Vicki Allison

Carol Beyer

Jordon Ott 

Marilyn Bruger

Deb Kempf

Cathy Schneider

Amy Kieke

Biostatistics

Burney Kieke, Jr.

Carla Rottscheit

Richard Berg

Risk Assessment

Frank Loge, Ph.D.

Elisabetta Lambertini

EPA

Angela Page

Cynthia Nolt-Helms

Shay Fout, Ph.D.

Phil Berger

Wisconsin WAHTER Study

Questions and Discussion
Wisconsin WAHTER Study

UV Intervention Effect on Adult 

and Children AGI (continued)

Treatment Tap Water Virus Concentration

(Mean total viruses in genomic copies/L)

Control Communities 1.316 

(n = 618)

UV Communities 0.326

(n = 585)

Immediately after UV 

Disinfection

0.032

(n = 191)

DIFFERENCE 1.022

Attributable Risk Percent estimated from virus difference = 6% to 14%

(Range depends on selecting adjusted or unadjusted models)
Wisconsin WAHTER Study

Permutation Test - Adults