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# Wednesday, March 16 8:00 a.m.–9:45 a.m. 

# Session V: Epidemiological Studies: What Does the Science Tell Us?110 U.S. EPA’s National Beach Conference



# Indicators of Water Quality and Health Effects at a Tropical and a Runoff Impacted Beach

Tim Wade U.S. EPA Office of Research and Development



### Abstract Background: Studies conducted by EPA at beaches with nearby treated sewage dis

charges established associations between gastrointestinal (GI) illnesses among swimmers and measurements of fecal indicator bacteria, Enterococcus, and Bacteroidales (marine beaches only) measured by quantitative polymerase chain reaction (qPCR). In 2009, EPA conducted studies at one beach in a tropical climate (Puerto Rico) and a second beach influenced by “urban runoff” (South Carolina). The goal was to examine water quality and illnesses among swimmers. Methods: Beachgoers were enrolled and upon leaving answered questions regarding swimming exposures. Ten to twelve days later they were asked about new illness symptoms. Six water samples were collected three times a day and tested for fecal indicator bacteria. Results: At the Puerto Rico beach, 15,726 individuals were enrolled. Swimmers reported higher rates of rash. Densities of fecal indicator bacteria were low and no single day exceeded EPA criteria for Enterococcus . In addition, the qPCR assay showed evidence of interference or inhibition. Consistent health relationships between fecal indicator organisms and swim

ming-associated illness were not established. At the South Carolina beach, 11,159 individu

als were enrolled. Swimmers reported higher rates of rash, GI illness, and earache. Only one day exceeded EPA criteria for Enterococcus

. Consistent health relationships between fecal indicator organisms and swimming-associated illness were also not established. Conclusions: At a tropical beach and a beach impacted by urban runoff, associations between illnesses among swimmers and water quality were not established. Possible explana

tions include better than expected water qual

ity, qPCR interference or inhibition, and lack of human fecal contamination. This is an abstract of a proposed presen

tation and does not necessarily represent EPA policy.



### Biosketch Dr. Tim Wade is an epidemiologist with EPA’s Office of Research and Development (ORD), Human Studies Division, in Chapel Hill, North Carolina. He received his Master of Public Health in Epidemiology and Biostatistics in 1998 from the University of California, Berkeley, and his PhD in Epidemiology in 2002, also from the University of California, Berkeley. He joined EPA ORD in 2003 as a post doc, and was recently named Chief of the Epidemiology Branch. His research focuses on quantifying and measuring the health effects of waterborne contaminants. Dr. Wade holds an adjunct appointment in the Epidemiology Division at the University of North Carolina.111 Day Two: Session V112 U.S. EPA’s National Beach Conference113 Day Two: Session V114 U.S. EPA’s National Beach Conference115 Day Two: Session V116 U.S. EPA’s National Beach Conference



# Rapid Indicators for Enterococcus and the Risk of Illness after Exposure to Urban Runoff Contaminated Marine Water: Results from a “Natural Experiment”

Presenter: Jack Colford University of California, Berkeley, School of Public Health 

### Authors: Jack Colford, Ken Schiff, John Griffith, Vince Yau, Catherine Wright, Benjamin Arnold, Joshua Gruber, Tim Wade, Charles McGee, Mark Gold, Steve Weisberg



### Abstract Traditional fecal indicator bacteria (FIB) culture methods are too slow to provide timely warnings to swimmers of potential health risks. We assessed the relationship between swim

ming-related illnesses and both traditional and new, rapid methods of detection using qPCR for Enterococcus species. We measured health at baseline and 2 weeks later in 9,525 partici

pants at Doheny Beach, California in 2007/08. Outcomes were compared for swimmers vs. non-swimmers. Among swimmers we com

pared associations of health to levels of indica

tors, including both traditional ( Enterococcus by EPA Method 1600 or EnterolertTM, fecal coliforms, and total coliforms) and rapid ( Enterococcus Taqman qPCR, Scorpion 1 qPCR, Scorpion 2 qPCR) methods. Beach conditions provided a natural experiment—the primary source of beach pollution was San Juan Creek, which flowed untreated into the ocean for weeks following rainfall, but did not flow when a sand berm formed across the creek mouth. This allowed us to study the impact of mark

edly different water quality conditions on the associations of traditional and rapid FIB mea

surements with health. We observed significant increases in gastrointestinal illness in swim

mers compared to non-swimmers, independent of the berm. Increasing levels of water exposure (body immersion/head emersion/swallowing) were associated with increasing risks of illness. When the berm was open (poor water quality), we observed significant associations between illness and both traditional and rapid methods for Enterococcus, but not when the berm was closed. These results suggest that the associa

tion of water quality indicators with illness is influenced by the conditions under which the indicators are applied.



### Biosketch Dr. Jack Colford is a Professor of Epidemiology at the University of California (UC), Berkeley, School of Public Health. He is a graduate of the Johns Hopkins School of Medicine (MD, 1985) and the UC Berkeley School of Public Health (PhD, Epidemiology, 1996). He completed a residency in internal medicine and a fellowship in infectious diseases at UC, San Francisco. He was Chief Medical Resident at Stanford University Hospital and completed board-certification in both inter

nal medicine and infectious diseases. He is the sole instructor in semester-long courses in advanced epidemiologic methods, interven

tion trial design, and meta-analysis, and was selected for multiple teaching awards at UC San Francisco, Stanford, and UC Berkeley by faculty and students. Dr. Colford has authored 70 peer-reviewed scientific publications, includ

ing numerous articles on waterborne diseases. He has been the principal investigator of four triple-blinded, randomized controlled trials of drinking water and health effects funded by the National Institutes of Health, the U.S. Centers for Disease Control and Prevention, EPA, and the UC. He collaborated with the World Bank on a comprehensive review evaluating the117 Day Two: Session V

effectiveness of drinking water treatments in the developing world. He was also the principal investigator of numerous observational studies focused on waterborne disease risks. Currently, he is the epidemiologist for a suite of water and sanitation interventions in India, South Africa, Peru, Tanzania, and Vietnam supported by the World Bank and the Gates Foundation. He is the principal investigator of a new award (September 2009) from the Gates Foundation for a 5-year, 2-country (Bangladesh and Kenya), 8-arm randomized controlled trial evaluating the single and combined benefits of water, sani

tation, and hygiene interventions.118 U.S. EPA’s National Beach Conference119 Day Two: Session V120 U.S. EPA’s National Beach Conference121 Day Two: Session V122 U.S. EPA’s National Beach Conference



# The BEACHES Study 1: Health Effects from Non-point Source Microbial Contaminants in Subtropical Recreational Marine Waters

Presenter: Lora Fleming University of Miami 

### Authors: Lora Fleming, Jay Fleisher, Jonathan Kish



### Abstract Background: The use of microbial water quality indicators to regulate recreational water bodies, particularly in sub/tropical recreational marine waters without known sources of sew

age, has been questioned. The objectives of this study were to evaluate the risks to humans from exposure to subtropical recreational marine waters with no known point source. Methods: 1,303 adult regular bathers were randomly assigned to bather and non-bather groups, with subsequent follow-up for reported illness, in conjunction with extensive environ

mental sampling of fecal indicator bacteria (FIB) and other indicator organisms. Results: Bathers were 1.76 times more likely to report: gastrointestinal illness (95% confidence interval = 0.94–3.30); 4.46 (0.99–20.90) for acute febrile respiratory illness; and 5.91(2.76–12.63) for a skin illness relative to nonbathers. Among the bathers, a dose-response relationship for skin illness was positively related to enterococci enumeration by mem

brane filtration (1.46 [0.97–2.21] per increasing log10 unit of enterococci) and positively related to 24-hour antecedent rain fall (1.04 [1.01–1.07] per increasing millimeters of rain). Acute febrile respiratory illness was inversely related to water temperature (0.74 [0.56–0.98] per increas

ing degree of water temperature). There were no significant dose response relationships between report of human illness and any other FIB or environmental measures. Conclusions: Bathers may be at increased risk of several illnesses relative to non-bathers, even in the absence of any known source of domestic sewage impacting the recreational marine waters. There was no dose-response relationship between gastrointestinal illness and increasing exposure to enterococci, even though this is the major outcome illness for many current water monitoring standards.



### Biosketch Dr. Lora Fleming is the Director of the European Centre of Environment and Human Health at the Peninsula College of Medicine and Dentistry in Cornwall, England. She was the former Co-Director of the National Science Foundation/National Institute of Environmental Health Services University of Miami Oceans and Human Health Center. Dr. Fleming received her AB (1978) and MD-MPH (1984) from Harvard University; her MSc (1979) in the History of Science from Imperial College (London University); and her PhD (1997) in Epidemiology from Yale University. She is a board certified Occupational and Environmental Medicine Physician and Epidemiologist. She works in the areas of The Blue Gym (a campaign and research program to facilitate improved awareness and recreational use of marine waters), marine and freshwa

ter toxins, and coastal pathogens, as well as research and teaching in Occupational and Environmental Medicine and Epidemiology. Dr. Fleming has published 190 peer-reviewed journal papers and book chapters, as well as many conference proceedings, books, and technical reports. She has also presented her research extensively in scientific and other
venues.123 Day Two: Session V124 U.S. EPA’s National Beach Conference125 Day Two: Session V126 U.S. EPA’s National Beach Conference127 Day Two: Session V128 U.S. EPA’s National Beach Conference129 Day Two: Session V130 U.S. EPA’s National Beach Conference131 Day Two: Session V132 U.S. EPA’s National Beach Conference



# Comparing Swimming-related Illness Associations with Fecal Indicators Quantified by qPCR and Culture-based Methods at a Freshwater Beach

Presenter: Jiyoung Lee Ohio State University 

### Authors: Jiyoung Lee, Jason Marion, Chang Soo Lee, Timothy Buckley



### Abstract Inland waters provide recreational benefits to vast portions of the U.S. popula

tion. Furthermore, with much attention being directed toward rapid indicators of water quality, inland water bodies have not been well represented. In this study, we engaged in an inland Ohio beach cohort study examining rela

tionships between fecal indicators and illness. Human health data and culture-based E. coli densities were obtained across 26 weekend days during the 2009 swimming season. Adverse health outcomes were reported via phone survey 8–9 days post-exposure. Complete data during the 23-day period was obtained from 692 individuals. Multiple fecal indicators were also measured via qPCR targeting enterococci and other markers ( Bacteroides-Prevotella group, B. fragilis group, B. fragilis-specific , and tetQ ). The strength of association between gastrointestinal (GI) and highly credible gastrointestinal (HCGI) illness for this population was determined via logistic regression adjusting for age, gender, food at the beach, reservoir inflow and time spent at the beach. The culture-based E. coli levels provided the only significant illness asso

ciation (highest E. coliquartile group adjusted odds ratio [AOR] = 5.14; 95% confidence interval [CI] 1.04–25.5). A marginal association (p = 0.08) was observed in this quartile with HCGI (AOR = 4.11; 95% CI 0.83–20.5). Despite lack of illness associations, enterococci and BacteroidesPrevotella markers were correlated (r = 0.474, p = 0.022; r = 0.444, p = 0.034) with culture-based E. colimethods, respectively. Overall, the water quality of the study site was good and the study population was small. Given these limitations, the traditional E. colimethod, albeit not rapid, was a better indicator of GI illness at this fresh

water beach.



### Biosketch Dr. Jiyoung Lee is an Assistant Professor of Environmental Microbiology in the College of Public Health, Division of Environmental Health Sciences at the Ohio State University. She received her BS and MS in Microbiology from Seoul National University, and her PhD in Environmental Health Sciences from University of Michigan. She has worked on beach fecal contamination problems, especially rapid detec

tion and contamination sources for the past 10 years. She has been a faculty member of the Ohio State for over 2 years and also has a dual appointment at the Department of Food Science and Technology. Her main research interests are (1) pathways of pathogen transmission in water and food, (2) new tools for understanding integrative health risks recreational waterborne illness, and (3) the impact of climate change on infectious agents.133 Day Two: Session V134 U.S. EPA’s National Beach Conference135 Day Two: Session V136 U.S. EPA’s National Beach Conference137 Day Two: Session V138 U.S. EPA’s National Beach Conference139 Day Two: Session VU.S. EPA’s National Beach Conference

140 

### Question & Answer Session

Question 1 Ali Boehm: One thing that seems consistent among urban runoff and nonpoint source studies is the importance of including a so-called “event modifier.” Going back to the Haille study in 1999, they used distance from the discharge point; in Jack’s [Colford’s] study, it was berm-open, berm-closed; in Laura’s [Fleming’s] study, they considered rainfall and tide to some extent. But your study didn’t include any event modifiers. I was wondering if you plan to include an environmental scientist or someone who has more physical knowledge of your urban runoff site in South Carolina to try and consider some event modifiers that might help you better explain your health data. I would just say it seems like a lost opportunity if you don’t do that.
Answer 1 Tim Wade: Absolutely, we didn’t do that. Although we didn’t, I’m sure someone with more expertise in that could specifically identify what the critical event modifiers would be. We did not necessarily have a berm that was either open or closed that lent itself to a natural experiment type of study. So, that’s a good suggestion. Lora Fleming: I just want to add that I was surprised by how little the physical parameters actually affected. I mean, we only found one positive association, and it was pretty tiny.
Question 2 David Turbow: Two questions for Dr. Fleming. First, in the study site, you were saying that there was no known point source of sewage contamination. Given that there’s an outfall pipe near there, how many million gallons per day [mgd] of treated wastewaters is discharged? And second, how long has that pipe been in place?
Answer 2 Lora Fleming: I don’t know if I can answer all of this, but Helena [Dr. Solo Gabriele] is here, so hopefully she’ll help. The pipe actually goes out 4 miles, and we’ve sampled out along that. Also, we’ve looked at the currents and, as far as we can tell, there’s no impact of that particu

lar site on our beach.
Question 2 (follow-up) David Turbow: How many million gallons of sewage are discharged into those waters and how long has that pipe been there?
Comment 2 Helena Solo-Gabriele: I have rough estimates, in the order of hundreds of mgd. But there have been expensive studies by NOAA that have shown that that water goes out into the Gulf Stream. Comment 2 (follow-up) David Turbow: Given that hundreds of mgd are discharged into waters within 5 miles of your study site, I think it’s a major threat to the internal validity of your study to claim that there’s no known source of sewage there. Comment 2 (follow-up) Lora Fleming: Can I just disagree a little bit? All our intensive monitoring has shown that if I want to make the Enterococcus levels go up, I go up to the swash zone and I sample there at high tide. That has nothing to do with the sewage source that’s several miles away and is taken away by the Gulf Stream. However, I haven’t done tracer studies, and your point is well-taken.141 Day Two: Session V

Question 3 John Heffelfinger: I have two administrative, non-science-based, questions for you. For all the panel

ists, can you give us a general sense of how much these [epidemiology] studies cost? I just have no idea of the scope of these things, dollar amount. Of course, there’s a lot of in-kind grad student resources involved, too. So, can each of the panelists quickly address that first?
Answer 3 Tim Wade: Our [EPA] studies, being some of the largest and most intense, run over a million dollars per beach site, including all the water sampling and testing. Jack Colford: I’d guess, all-in-all, about $1.5 million total. Lora Fleming: We did ours on a shoestring, which I wouldn’t recommend to anyone; it was about $600,000. Jiyoung Lee: $250,000 to $300,000.
Question 4 John Heffelfinger: Okay, great, thanks. And then kind of a bureaucratic question, maybe particularly for Tim [Wade]. Were any kinds of pre-approvals or clearances required for these studies? I’m thinking in particular the Federal Paperwork Reduction Act requirement; what were your experiences there?
Answer 4 Tim Wade: We have a standing Office of Management and Budget (OMB) approval, which I don’t believe non-federal people need to get. So that was an additional burden for us, but we have an OMB approval under the Paperwork Reduction Act. And then there are human subject approvals that we have to run through ethical boards. Does anyone else want to add anything?
Comment 4 Lora Fleming: One issue for point source areas in terms of doing a randomized trial like we did is that human ethics boards probably will not allow you to do it. So, we were able to do it because it was a nonpoint source and because we used only people who regularly bathed there. Jack Colford: We didn’t go down that path because we’re different in California and we were fairly certain that our human subjects board wouldn’t consider that. Tim Wade: I’m not so sure they wouldn’t approve it, because certainly other, more invasive, randomized things are approved, like in fields of medicine. But I agree it would raise other issues. Especially if you knew, or had a strong idea of, what was in the water at the beach— like if you knew there were human pathogens at the beach that you were intentionally exposing to people, it’d probably come under a lot higher scrutiny, particularly for children.
Question 5 (Unknown): I have two questions. I thought it was very interesting to be able to compare Puerto Rico to Miami. What I found interesting about both Puerto Rico and also Miami is that skin and respiratory illness came up. I was wondering the opinion of whether this is something that needs to be looked at further? Is EPA considering non-GI endpoints?
Answer 5 Tim Wade: Yes, increased skin rash among the swimmers is something that we see—I think Jack [Colford] has seen and even written a review paper on this—it’s commonly seen. And I’ll be interested to see what Lora [Fleming] has to say about it, but I think the struggle there is the causal mechanism for it. The nice thing about the GI is that there’s at least a theoreti

cal, causal basis that I laid out in the first couple of slides. Again, going back to the wholeU.S. EPA’s National Beach Conference

142 paradigm of the human fecal source that they are marking GI pathogens, but with the skin rashes—I think it’s a bigger struggle to make that causal link. You discussed it, at least in your paper, I think.
Comment 5 Lora Fleming: One of my problems with the skin is that it’s totally self-reported. If I had a lot of money, I would have had a dermatologist looking at them. Also, another factor might be in tropical areas, heat-related rash kind of thing as well. And the other thing that Dr. Lee mentioned is that there are other organisms out there, and HABs [harmful algal blooms]; for example, copepods, and things that we weren’t looking at, and don’t traditionally look at. I agree with you, I think that models need to incorporate those much more and not just be focused on the endless loop of Enterococcus

. Question 6 (Unknown): But it was interesting to see that both tropical and subtropical studies showed that, which is different than the other sites. My second question is related to Ali’s [Boehm’s] question. With the other studies, whether physical/chemical parameters were looked at. I was interested in knowing, Tim [Wade], in the two studies that you conducted whether that was looked at, and also Dr. Lee, in your study as well.
Answer 6 Tim Wade: We do look at those things and we’ll admit, maybe not to the extent that we could have. But certainly, we look at things like rainfall, turbidity, temperature—and basically, they almost never show a good relationship on their own with illness. And I don’t recall specifi

cally at Surfside Beach, we were in a bit of a rush to get that report out. Jack Colford: I’ll just add, in the next session, Vince Chow, a doctoral student at [UC] Berkeley is going to present a talk with data from Avalon [Beach], where Ali Boehm from Stanford collected a lot of these physical indicators. Vince is working on methods to incorporate those into the analyses. Jiyoung Lee: We also compared water quality and E. coliand other genetic markers. We saw some really significant correlation, especially phosphorus and E. coli . Jason [Marion] is presenting a poster about that. Based on phosphorus and E. coli , can you predict tomorrow’s water quality? In other words, based on yesterday’s phosphorus and E. coli , can we predict today’s E. colilevel for advisory purposes? That is another presentation. Based on our princi

pal component analysis, we saw a significant correlation between water inflow and E. coli

— especially yesterday’s inflow. With precipitation, rainfall, and E. coli , we saw a correlation. For viruses, we saw the opposite. So for viruses, when there was very low inflow, we saw more viruses. Thus, it looks like a viruses and E. colimight have a different input transmis

sion routes or input sources.
Question 7 Lyman Welch: I have a question about sensitive populations and I was wondering why, Dr. Fleming, you didn’t include children in your study? But, did you look at the elderly or pregnant women to see if there’s any increased sensitivity there. And then, Dr. Lee, it looked like you included children, I wasn’t sure if you looked at children specifically to see if there was any greater connection or sensitivity to that population or to other potentially sensitive populations?
Answer 7 Lora Fleming: I wish I had included children. I made a decision not to do that for two reasons; one issue was just logistically—we had to do a lot in a very little time. Children are a bigger issue for us because how would you get a child to get their own water sample? There were also issues of how we were logistically going to play out the study design with children. The143 Day Two: Session V

other issue was that our IRB [internal review board] indicated that they were going to give me a big hassle about it, and I needed to start the study. But having said that, I was part of the early [pilot study] group who said that children are probably the most important target group in terms of sensitivity. We took age into account and that was not an issue. I think there were only two or three pregnant women, and you can’t say anything about that. It’s just not enough statistically to look at. Jiyoung Lee: Our sample size is so small that we don’t see a significant difference in terms of age. Lora Fleming: Except that you have a lot of stuff about diapers and eating. And I’ll bet you if you look at the kids, the diapers, the eating, there’s a real cluster. Jiyoung Lee: Yes, exactly. The consumption of food is really a confounder, especially in the children.
Question 8 Bernard Franklin: Lora [Fleming], you had indicated in your study that you saw higher incidence at high tide. Could you comment on potential re-growth?
Answer 8 Lora Fleming: I’ll probably punt that over to Helena [Dr. Solo-Gabriele]. And remember, it was higher incidence of the Enterococcus ; it did not play out when we did the epi study. Helena Solo-Gabriele: We’ve conducted some preliminary studies on that sand. And the sand can dry for a period, and if you re-wet it, you can increase the number. But you don’t know if it’s re-growth, but there is a spike in culture.
Question 9 John [unknown last name]: I’m very familiar with Tim’s [Wade’s] studies. Jack [Colford], this is for you. It looks like you have very much of a binary situation with the berm-open, berm-closed, and I was just wondering—the extent that the human contribution is, obviously, a big variable here—if there is any effort to characterize the discharge from that waterbody in terms of [inaudible]?
Answer 9 Jack Colford: Great question. I’m going to turn to another John, Dr. John Griffith [SCCWRP] to comment on some of source tracking and other sort-of parallel studies that are going on in concert. John Griffith: There were several markers run. The data’s still out on some of them, although Jack didn’t show that data this time. Polyomavirus did pop up as significant correlation when the berm was open, but not when the berm was closed. When the berm was closed, we didn’t see anything as far as a correlation. We still have to run quite a bit of the data.
Question 10 Dianne Waters: Two questions. First, I noticed with the epidemiology, most of the reporting had demo

graphics, it’s 95 percent white and I think another one was 92 percent white. I was wondering if there are any plans to account for that? I noticed one is reporting as a certain percentage as Hispanic—I think the one in Puerto Rico. However Hispanic is not a race, so you can’t really compare that demo

graphic to the demographics other places and try and draw conclusions. My second question is that I noticed that there was an effort to take into account nonpoint sources, and most of them were over land. Has there been any effort to take into account stormwater runoff when it’s coming from stormwa

ter outfall pipes?U.S. EPA’s National Beach Conference

144 Answer 10 Tim Wade: Right, Hispanic is an ethnicity and we did clump them together because despite the OMB requiring us to do it, the people actually often insisted that they wouldn’t give a race after saying Hispanic. Since it was a very sensitive thing, we did group them like that. And the beach that we studied in South Carolina wasn’t typical, I may have said it was, but that was a mistake. I think that we had a more mixed population at some of our other beaches in terms of racial makeup. Our goal with the study was to reflect the beach going population, so whoever was there. And stormwater, I’m not sure about that. The best study I know that directly linked to stormwater was the Haille study that was mentioned before, that looked at storm drains, but I don’t think that any of our studies addressed that.
Question 10 (follow-up) Dianne Water: Most of the studies that were considering nonpoint source pollution were only consid

ering a swash or some sort of over-land flow. And technically stormwater is considered a point source, but it’s sort of a nonpoint source that gets collected in the pipe and is collected as a point source at the end. Were there any plans to consider whether there are different things going on when you have water flowing underground and discharging from a pipe that’s nonpoint source, point source pollution, rather than just over-land flow? Answer 10 (follow-up) Tim Wade: Right, we don’t have any plans for that.
Comment 10 Lora Fleming: Could I just comment on race/ethnicity too? I have similar experience in Miami with the fact that most Hispanics consider themselves Hispanic rather than identifying with a race. Also, in Miami, there is a long history of a so-called “colored beach,” where it was a segregated beach, it’s actually on this same island, it’s been turned into a historic site, but many of the black population of Miami do not swim because of that—because they had terrible currents and people used to drown there regularly. In terms of our study, there were black people in the study, but the majority are from island communities, not African American.
Question 11 Mark Gold: Tim [Wade], sort of a follow-up. On the Haille study, there was a pretty strong associa

tion on one particular beach in proximity to swimming next to a flowing storm drain. So this sort of follows up with Ali’s [Boehm’s] point too. It looked like you had that opportunity to look at that data set in South Carolina in that manner, because of that swash, having a flowing drain right there. Have you taken a look at that to see what the instance of illness is? Fifty yards, 100 yards, that kind of thing?
Answer 11 Tim Wade: Good point. We haven’t done that yet. But I definitely do see that as something that we could do. We have the locations of where people were in relation to where, presum

ably, they swam the most. We have GPS measurements of where they were, at least at the time of the interview. So, we haven’t done it yet, but that’s an example of something else we could do with the data.
Question 11 (follow-up) Mark Gold: And one other thing on the South Carolina study is that some of these epidemiology stud

ies, it’s amazing that when it’s announced that you’re going to move forward and do the epidemiology study, this sort of phenomenon of improved water quality occurs. We’ve seen it in New Jersey, we’ve seen it in Mission Bay, and we’ve seen it in a bunch of different places. Was there any kind of con

certed effort by the community, for example, to do source abatement prior to this study? Infrastructure improvements, that kind of thing?145 Day Two: Session V

Answer 11 (follow-up) Tim Wade: It seems to happen all too commonly with epidemiology studies; if you want to clean up a place, do an epi study there. Sometimes I’ve actually suspected people actually cleaning it up. But I didn’t have any indication that anything like that was going on, not that I know of.
Question 12 Gil Dichter: Just to follow up on that question to the subtropical testing for the epi studies, since the data didn’t really show any correlation between the qPCR and illness, how are we going to use the data to support either certain levels for enterococci or E. coli against illness? Do we plan to do some addi

tional studies in subtropic waters to get correlation data? This takes me back again to the 1986 study, where many epidemiologists disagreed with the data. Are we going to have this again?
Answer 12 Tim Wade: Probably. I don’t know if that was directed at our study; we did not see an associa

tion with the qPCR-based indicators. There were some measurement issues and how that plays out, I’m not sure. But I understand the point.
Question 13 Stefan Wuertz: I have a general question to all the presenters. How did you deal with nondetect data? I think this is probably not so important to the standard cultivation-based methods, but it certainly is very relevant for qPCR data, in particular pathogens, and if you then do correlations or regression analysis. So I just want to ask how you dealt with nondetects and whether you included detection limits?
Answer 13 Tim Wade: This was brought up by some in our internal peer review regularly, so we kind of did an exhaustive—especially with the data that’s already been published—process of vari

ous types of approaches to statistically handle it. I think that we mention in our publications that basically, the health relationships are really unaltered by the approaches used. Now, I only feel comfortable doing this when you are under the, maybe 15 percent nondetects; when you start getting up to 50, 60, 70 percent nondetects, I think it’s a real statistical issue that no one agrees on the best way to use the data. So I think there some further statistical work that needs to be done—especially with qPCR data, how to handle it. Jack Colford: Yes, I think it’s an important sort of sensitivity analysis to do. And John [Griffith], correct me if I’m wrong, did we assign half the detection limit to the nondetects? But we were following in Tim’s [Wade’s] footsteps, because you had tried some maximum likelihood approaches. Tim Wade: Well, we did many approaches and what we actually found was that, in terms of establishing a health relationship, using half was just as good as using some non-parametric statistics and maximum likelihood approaches, so we ended up just using half, but we had a sensitivity analysis that showed that we could justify it in other...
Question 13 (follow-up) Stefan Wuertz: So you used a substitution method, which is actually usually not recommended? Answer 13 (follow-up) Tim Wade: It’s not recommended for describing the level, but in terms of actually developing association with health effects, from my understanding, it’s not as clear.U.S. EPA’s National Beach Conference

146 Question 14 Elizabeth Halliday: My question is about the negative controls that were used in these studies. I think it’s really interesting that in the Miami study, you really isolated the bathers and the non-bathers; like the non-bathers had no interaction with the beach and a picture tells a thousand words with themsitting on the tarp and plastic chairs. I wonder about, especially at Doheny [Beach], whether the nonswimmers still had significant interaction with the beach and beach sands and whether you think that might affect the odds ratio between the swimmers and non-swimmers?
Answer 14 Jack Colford: Let me clarify: I didn’t make a big point of this in the talk, but the swimmers versus non-swimmer analysis obviously includes the non-swimmers. The other analyses for the indicators, it’s only the trend among the swimmers. It doesn’t affect it much when we use the non-swimmers, but they’re done separately. We didn’t see it having much of an impact when you include them. The field has gone back and forth on this; there have even been pub

lished papers on the use of non-swimmers in these analyses. So we’re trying to be consistent with what EPA has done.
Comment 14 Tim Wade: Just to add to that by using swimmers as your own control—there’s two compari

sons: “versus non-swimmers” and “among swimmers;” and “among swimmers” is a bit more rigorous because the swimmers would also not have this recall issue, which Lora [Fleming] notes is still an issue in a randomized study. So, presumably, they would have the same recall bias as the other swimmers. Another advantage of developing relationships among swimmers is that they are probably effectively blind to what they’re exposed to, their level of indicator. Comment 14 (follow-up) Lora Fleming: Regardless of the study design? Tim Wade: Regardless of the study design, right. I think the randomized design does give you a much better idea of what the non-swimmer rate is, if you can sort out the recall issue. Lora Fleming: And the problem with the randomized design is that you effectively eliminate the ability to look at dose-response within the swimmers because you made them all have the same exposure.
Question 15 Sharon Nappier: Two quick questions for Dr. Lee. For the first one, you found associations with the E. coli culture in the GI, not for E. coli qPCR. I didn’t know if you could speak to the frozen filters and if you did some recovery analyses, and how they were stored and handled? Also, you have associations with adenovirus, and I didn’t know if you plan to look at norovirus specifically?
Answer 15 Jiyoung Lee: I think that kind of study has been done by other researchers. The beauty of qPCR is that their DNA should stay in the water. So actually, we measured E. coliusing plate-based methods and the Enterococcus for qPCR. So the DNA should remain pretty much in the sample, and then the gene copies numbers are a lot in the frozen samples. So, we didn’t really do the decay of frozen DNA as time went by, but, based upon the literature, or other researchers, I think that should be okay. But, I think the freezing temperature is more significant; so rather than –20 °C, –80 °C is much better. So, I think that the temperature [might] affect some DNA decay, but the duration should be okay. We did norovirus first; that’s why I collaborated with Dr. Linda Safe, who is the expert. We didn’t find any norovirus. We only targeted human noro

virus, and the norovirus was not there. And then also, we compared our measurement with the Linda Safe group, and we found similar results. We found in addition and then concentra

tion procedure, we paralleled our study, but we found the same. So I think that our sample water volume was too small, so that’s why we didn’t see norovirus.