Wisconsin WAHTER Study 

# Wisconsin Water And Health Trial for Enteric Risks (WAHTER Study) Part 1: Risk of Illness from Municipal Groundwater Consumption Mark Borchardt, PhDWisconsin 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, 1998Wisconsin 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 risksWisconsin 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 - 58Wisconsin 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 GroundwaterWisconsin 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 mWisconsin 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 - MadisonWisconsin 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 PeriodsWisconsin 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 Non Intervention 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

Service DesignUV dose = 50 mJ/cm2Wisconsin 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 weekWisconsin 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 periodWisconsin WAHTER Study 

# Participating Households’ Characteristics

• Beginning enrollment: 621 households
• Ending enrollment: 440 householdsWisconsin WAHTER Study 

# Study Subjects’ Characteristics

• Beginning enrollment: 1,079 children, 580 adults
• Ending enrollment: 765 children, 413 adultsWisconsin 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 tapsWisconsin WAHTER Study 

# Virus Captured by Glass Wool Filtration Volume sampled (liters) Mean = 860 Range: 76 – 2,067 N = 1,786Wisconsin 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 sequencingWisconsin WAHTER Study 

## Determine the association between tap water virus concentrations and community illness rates 

# Objective 1Wisconsin WAHTER Study 

## Virus Types, Frequencies, and Concentrations in Distribution Systems

N = 1,204 samplesWisconsin 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 elevatedWisconsin 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 = 2.2Wisconsin WAHTER Study 

### All ages Adjusted for community and period 

# Total Virus Concentration in Tap Water and 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 Virus
Positive, and Maximum Concentration

P - values < 0.1 < 0.05 < 0.01 < 0.001Wisconsin WAHTER Study 

### All ages Unadjusted 

# G1 Norovirus Concentration in Tap Water and AGI Incidence

Virus conc for significant rate ratio = 0.001 P - value = 0.0005Wisconsin WAHTER Study

0 20 40 60 80 100 120

Spring ’06 Fall ’06 Spring ’07 Fall ’07 Study Period Adenovirus Enterovirus GI Norovirus Hepatitis ANumber of Positive Samples 

# Virus Types Detected in the Distribution SystemsWisconsin 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 4Number of SamplesWisconsin WAHTER StudyWisconsin 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 = 0.6Wisconsin 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 sequenceNumber 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 0Wisconsin WAHTER Study

0 20 40 60 80 100 120

1 2 3 Wells Post - UVNo. of Samples ICC-PCR PositiveNo. of Samples qPCR Positive

Distribution SystemWisconsin 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 riskWisconsin 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 UVWisconsin 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 I iC is the illness incidence in the ith community during the control period and I iI is the illness incidence in the ith community during the intervention period. 

### • H 0 = the true difference in AGI incidence between intervention and control periods is 0. 

### • Test H 0 using the permutation test 

### ( **) **  ** **  ** **  ****

**−**



### ∑

**=** 14

1 14

1 i iI iC 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’ wellsWisconsin 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/person year attributed to groundwater
• 2,200 times higher than EPA acceptable riskWisconsin 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 interventionWisconsin WAHTER Study 

## Power Analysis for Overall Comparison of Intervention Versus ControlWisconsin 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 interventionWisconsin 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 = 0.8Wisconsin 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 Virus
Positive, and Maximum Concentration

P - values < 0.1 < 0.05 < 0.01 < 0.001

## Period 1 Only Attributable Risk for Groundwater
borne AGI

All analyses weighted by sample size (i.e., person - time) *Adjusted for age, gender, day care attendance, and virus concentrations in the communities’ wellsWisconsin 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 interventionWisconsin WAHTER Study 

## Total Virus Concentration and AGI Incidence by UV Treatment StatusWisconsin 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 proceduresWisconsin 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 ContaminationTotal Viruses Genomic Copies/L Sample Location Number Samples Mean Median 75 th Percentile 95 th 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 1Wisconsin WAHTER Study 

# Virus Concentrations from Source to TapEvent 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 SystemsWisconsin WAHTER Study 

# Thoughts, Opinions, Gut Feelings

• There is a significant public health risk from drinking non disinfected 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 TeamWisconsin 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 BergerWisconsin WAHTER Study 

# Questions and DiscussionWisconsin 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