### 1 

## F INAL 

## R EPORT

T HE 

### P ILOT 

### E PIDEMIOLOGIC 

### A SSESSMENT OF 

### M ICROBIAL 

### I NDICATORS

FOR


### M ONITORING 

### R ECREATIONAL 

### W ATER 

### Q UALITY

I N


### M ARINE 

### S UB 

### /T ROPICAL 

### E NVIRONMENTS

Principal Investigators : Lora E Fleming MD PhD MPH MSc, (University of Miami)

Helena Solo Gab riele PhD (University of Miami) Jay M. Fleisher P hD (Nova Southeastern University) Samir Elmir PhD PE (Miami Dade County Public Health Department) Chris Sinigalliano PhD (NOAA Southeast Miami) Lisa Plano MD PhD (University of Miami)

John Wang PhD (Universit y of Miami)

Collaborators University of Miami: Mr Jonathan Kish MPH, T omoyuki Shibata PhD, Amir Abdelzaher MS, Gu o qing He PhD

, Mary Wright MS

, Julie Hollenbeck BA

NOAA Southeast Miami Lab: Kelly Goodwin PhD

, Maribeth Gidley DO MPH, David Wanless MS

Mi ami

- Dade Dept. of Health: Gary Miller Centers for Disease Control (CDC): Lorrie Backer PhD University of Florida: Carol Palmer PhD, Fred Bonilla PhD , To nya Bonilla PhD

BCS Laboratories: Troy Scott PhD , George Lukasik PhD University of South Florida: Vale rie Harwo od PhD , Shannon McQuaig MS

NOAA Charleston : Jill Stewart PhD , Jerold Dickerson PhD

WHOI: Rebecca Gast PhD University of Missouri: Charles (Andy) Carson PhD , Helen Yampara

- Iquise PhD

### 2 A CKNOWLEDGEMENTS

**The researchers would like to dedi cate this**  **research to the**  **memory of Ms Seana Campbell, a very talented, hardworking and creative young researcher who enriched all people whose**  **lives she touched, and who died too young.**

The researchers would also like to thank the following University of

Miami and FIU Students , other student researchers, and other

researchers for their assistance in the performance of this study : Cristina Ortega (NSF REU), Kelly Withum, Anna Christy Garza

, Hasan Abdel Fattah, Karen Kajder, Matthew Straney, Michael Lara,

X iaofang Zhu, Reshma Ramoutar, Michael Schoor (NSF REU)

, Erik
Kumetz, Katie Herleman, Yasiel Hernandez, Rafael Hernandez (Howard Hughes Scholar)

, Maria Del Carmen Vazquez MD , Evelyn Davila, John Clark PhD (NIEHS F30)

, Paulo Pinheiro MD, Zacha ry Lipschultz, Sharde Chambers, Derek Schesser, Jakub Bartowiak

, Gloria

Scorzetti ; Florida Dept of Health: Erin O’Connell MPH , Pedro (Paco) Castellones MPH , Gary Miller, Racquel Mohammed, Michael Rybolowik, Gerard Philizaire, Erin May ; EPA Intern: Lauren Crowder ; NOAA H ollings

Scholar: Rene M. Boiteau

The researchers would also like to thank the following individuals and organizations for their support without which this study as well as our ongoing research would not have been possible: Dr Sharon Smith , Dr Peter

Minet te, and the NSF NIEHS Oceans and Human Health Center; SeaAquarium Staff and Facility; A merican Express; the University of Miami Rosenstiel School of Marine and Atmospheric Sciences ( RSMAS ) facilities, RSMAS security, RSMAS Marine Biology and Fisheries (MB F Division), and the Schmale and Fell laboratories,

the RSMAS Business O ffice , Aida Diaz and Sidney Hartley; Miami Dade Parks & Recreation s; Miami Dade County Dept of Health (Envi ronmental Health, Microbiology); Dr David Kay and his European collaborators; Florida Department of Health

, and the Grants Mana ger for this project, David Polk; University of Miami

- College of Engineering; University of Mi ami

– Miller School of Medicine; the NOAA AOML Laboratory ; Mote

Marine Laboratory: Kate Nierenberg MS.

T

his P ilot Epidemiology Proposal was funded in part from the following sources : the National Center for Environmental Health (NCEH), Centers for Disease Control and Prevention (CDC); Florida Dept of Health (FL DOH) through monies from the Florida Dept of Enviro nmental Protection (FL DEP); the Environmental Protection Agency (EPA) Internship Program; the National Science Foundation (NSF) and the National Institute of Environmental Health Sciences (NIEHS) Oceans and Human Health Center at the University of

Miami Rosenstiel School ( NSF 0CE0432368; NIEHS 1 P50 ES12736 ) and NSF REU in Oceans and Human

Health , and the NSF SGER ( NSF SGER 0743987 ) in Oceans and Human Health

. We would also like to thank IDEXX for their support of our project through the provision of sup plies needed for the chromogenic substrate analysis of enterococci.

### 3

### 4 A BSTRACT

Microbial water quality indicators are found in high concent rations in sewage, and are used to determine whether or not water is safe for recreational purposes. Recently, concerns have been raised about the appropriate use of microbial indicators to regulate recreational uses of water bodies. This is particularly (although not exclusively) true for water bodies located in sub/tropical environments with no known point

sourc es , due to the potential for microbial regrowth in sub/tropics, and the fact that studies used to set national guidelines were not conducted in sub/tropical environments nor with no known point sources . The primar y goal of this study was to evaluate the r elationship between human health, and the physical and microbial

characteris tics of non point source sub/tropical recreational marine waters and its shoreline. This goal was addressed by measuring human health effects and microbial water quality at a recr eational beach site withi n a sub/tropical climate in conjunction with the ongoing development of a predictive, coupled hydrodynamic and

microbial fate model that hopefully in the future can be used to distinguish impacts from sewage sources versus regrowth of microbes, each of these sources possibly resulting in different health effects as observed at

the beach site.

Substantial environmental pilot data had already been collected fo r the sub/tropical Study S ite chosen for this research. These pilot data, as well as the literature, indicate d that exposure to contaminated recreational marine waters may result in human health effects, and that the shoreline sediments were one likely source of

indicator micr o bes. In the Pilot Project, h uman health was evalua te d in the through a n epidemiologic study

which randomly assign ed volunteer adult participan ts (residents of south Florida who regularly bathed in recreational marine waters) to bather and non

- bather groups , with subsequent follow up for reported illness

. T his epidemiologic effort was conducted in conjunction with an ongoing e xtensive sampling of indicator organisms and pathogens as well as hydrodynamic modeling. The environmental assessment was conducte d

such that measures of indicato r organism exposures were assigned directly to each individual bather

, as well

as additional environmental measures

. Microbe measur ements include d traditional (enterococci, fecal coliform

, E.coli ) and non

- traditional ( C. perfringens

, Bacteroides ) microbial indicators, as well as direct measurement of microbial pathogens ( S. aureus , enterovirus, C. parvum , and G. lamblia ). Individual sample s were assayed for

enterococci and Staphylococcus aureus . Daily composite samples made from individual samples collected on each day were a ssayed for multiple fecal indicator microbes (enterococci, C. perfringens , fecal coliform, E. coli )

and waterborne pathogens (ent e rovirus, Cryptosporidium , and Giardia ), and S. aureus as a skin infection agent. In addition to these organisms, daily “speci al” samples were also assayed for enteric viruses, Cryptosporidium , and Giardia

. Environmental parameters were also measured throughout the study.

The study results indicated that there was an increased risk of reporting gastrointestinal symptoms with ba thing exposure at the non point source subtropical recreational marine water. Furthermore, there appeared to be an association between enterococcus (by membrane filtration method) levels and report of bather gastrointestinal symptoms at or above 40 CFU/10 0 mL of enterococcus based on an individual sample

. The Study Site, which

is a non

- point source of fecal contamination, showed intermittent high levels of indicator microbes in this study. Nevertheless, these intermittent high levels of indicator microbe s did not coincide consistently with the presence of these pathogens. Future studies should also focus on developing fate and transport models

, epidemiologic studies, and quantitative microbial risk assessments which incorporate human, microbiological

and oceanographic data.

I NTRODUCTION

The description of the methods and results below is focused on the P ilot Epidemiologic Project of the ongoing

NSF NIEHS Oceans and Human Health Center “Recreational Microbes” Study; the extensive environmental

monit oring and hydrodynamic modeling which occurred concomitantly with the epidemiologic study are currently funded by an ongoing Research Study of the NSF NIEHS Oceans and Human Health Center of the University of Miami with collab orators from NOAA, the CDC, th e Miami Dade County Dept of Health , University of Florida, University of South Florida, and other collaborators . This Pilot Epidemiologic Study provided for the collection of human epidemiologic data in conjunction with the ongoing already

- funded environm ental monitoring and hydrodynamic modeling, allowing for “leverage” of funding and resources. Ultimately, the

### 5 human, environmental and oceanographic data will be used to develop a predictive microbial fate and transport model with broad applicability to b eaches and recreational marine waters in the sub/tropics with appropriate modifications for local conditions. Model results will complement recreational water quality monitoring in the sub/tropical marine environment without a known point source thereby i mproving the likeliho od of reliable risk

assessment.

S TATEMENT OF P ROBLEM

Based on risk assessments from the World Health Organization (WHO) and academic research sources

, Shuval (2003) estimated that globally each year there are in excess of 120 millio n cases of gastrointestinal disease and in excess of 50 million cases of more severe respiratory diseases caused by swimming and bathing in wastewater

- polluted coastal waters. Dwight et al (2005) estimated that the economic burden per gastrointestinal ill ness (GI) was $36.58 while it was $77.76 per acute respiratory disease, $37.86 per ear ailment, and $27.31 per eye ailment (2001 values in Orange County , California). The authors then estimated the cumulative public health burden from the excess illnesses associated with coastal water pollution to be 3.3 million dollars/year from just 2 recreational marine beaches in California. Finally, Given et al (2006) used epidemiological dose

- response models to predict the risk of gastrointestinal illness at 28 beac hes spanning 160 km of coastline in Los Angeles and Orange Counties (California)

. They estimate d between 627,800 to
1,479,200 excess cases of gastrointestinal illnesses occur red at these beaches each year , corresponding to an

annual economic loss of 21 to 51 million dollars (in year 2000 dollars).

Recently, concerns have been raised about the appropriate use of microbial indicators to prevent illness and to regulate recreational uses of recreational marine water bodies, particularly true for water bodies located in sub/tropical environments and without known point sources , due to the potential for microbial regrowth in sub/tropics, and the fact that studies used to set national guidelines were not conducted in sub/tropical

environments , focused on location s with known point sources, lacked individual exposure information , and had epidemiologic methodologic weaknesses (Haile 1999, Wade 2003, Dwight 2004, Colford 2007, US EPA 2007, Yamahara 2007, Boehm in press)

.

S PECIFIC A IMS AND H YPOTHESES

The primary goal of the Pilot Epidemiologic S tudy was to conduct a prospective randomized epidemiologic study to assess the human health risks associated with swimming in sub/tropical seawater contaminated with infectious microorganisms ( while minimizing the significa nt bias seen in prior epidemiolo gical studies) in collaboration with ongoing intensive environmental monitoring and hydrodynamic modeling. The Pilot Epidemiological Study was designed to address 3 major methodological weaknesses inherent in previously pub lished epidemiological studies assessing the health effects of bathing in waters contaminated with domestic sewage. The se methodological weaknesses were : 1) Failure to control for the substantial amount of temporal and spatial variation in indicator organ ism density shown to occur within just a few hours and within several meters at marine bathing locations; 2) Failure to relate indicator organism density directly to the individual bather; and 3) Failure to rigorously control for non

- water

- related risk fac tors for the outcome illness under study. The design of

the Pilot Epidemiological S tudy addressed these weaknesses by controlling bather exposure, by individual study participant s monitoring of their own environmental exposures ( in dicator organism densiti es )

, by pre

- and post

exposure interviews with each study participant, and by randomization of exposure among only bather participants (Cabelli 1982, Dufour 1984, Calderon 1991, Haile 1999, Wade 2003, Kay 2004, Dwight 2004, Wade 2006, Boehm 2007, Colford 2 007, US EPA 2007, Yamahara 2007, Boehm in press). Furthermore, the S tudy was designed to evaluate the exposures of and health risks to bathers in sub/tropical recreational marine waters with no known point source of microbial pollution.

Specific Aims : **Th e objective**  **s of this Pilot Epidemiologic S**  **tudy were to begin to evaluate the**  **relationship between microbe densities and reported symptoms in human subjects with random assignment to recreational marine water exposure and intensive individual microbial moni**  **toring in**  **subtropical recreational marine waters with no known point source**

**.**

### 6 The hypotheses tested for the epidemiologic study were:

**H1 : People exposed to recreational waters contaminated with infectious microorganisms**  **would be**  **significantly more like ly to report symptoms than unexposed similar people who go to the**  **beach but do not go into the water.**

**H2 : The organism densities sampled at the time of ex**  **posure in the marine waters would be associated**  **with reported human symptoms.**

Ultimately, the human, environmental and oceanographic data will be used to develop a predictive model with broad applicability to beaches and recreational marine waters in the sub/tropics with appropriate modifications

for local conditions.

B ACKGROUND

The primar y significa nce of this Pilot S tudy was that it began to a ddress an important data gap. The use of indicator microbes for water quality monitoring in sub/tropical (tropical and sub

- tropical) environments has been questioned, in particular for sub/tropical coastal zon es where there are no k nown point sources of pollution (Haile 1999, Wade 2003, Colford 2007, US EPA 2007, Yamahara 2007, Boehm in press). The premise is that indicator microbes within these regions regrow due to climate conditions conducive for their regr owth, and are thus not representative of sewage sources of pathogens (Yamahara 2007) . The few epidemiologic studies

conducted previously in sub/tropic al environments (Fujioka 1994, Kueh 1995) showed no statistically significant relationship between human h ealth and enterococcus, the current ly recommended indicator microbe for evaluating marine water quality in the US (Haile 1999, Colford 2007) . Although these studies challenged the use of current methods for sub/tropical environments with non point sources , they did not establish a

substantial case for a new method or new set of indicators. Furthermore, t hese prior studies did not include an oceanographic component aimed at simulating the distribution and number of microbes in water during

different enviro nmental conditions.

The Pilot Epidemiologic Study discussed in this Report differed in that it was conducted in conjunction with coordinated ongoing interdisciplinary efforts, including intensive individual microbial exposure monitoring and

an oceanogra phic component to evaluate the distribution and inter

- relation of microbes, human illness and

other environmental factors ; furthermore, this study used a different epidemiologic methodology in which bathers were randomly assigned to exposure, and then coll ected their own environmental water exposure samples . The researchers in this Pilot Epidemiologic Project consider that this combination of interdisciplinary

efforts will ultimately build a unique and stronger case concerning which microbe or monitoring me thodology is the most effective for establishing human related illness in sub/tropical marine environments and non point sources in sub/tropical recreational marine waters.

**Human Health Data :**  Several epidemiologic studies have found that, compared with bathing in uncontaminated waters, bathing in temperate recreational waters contaminated with domestic sewage or storm

drain runoff (ie. known point sources) has been associated with an increased risk for transmission of infectious diseases, including gastr oenteritis, febrile respiratory illness, eye and ear infections, and skin infections and rashes. However, there were weaknesses in the designs of many of these studies, including the absence of

data capable of assigning individual estimates of exposure to each bather resulting in exposure

misclassification

, a failure to account for other risk factors for the illnesses of interest (including the possib ly inappropriate comparison of cohorts of bathers to cohorts of non

- bather s) , t he presence of a known point

source (rather than the more common scenario of no known point source) , and purely temperate not sub/tropical marine environment s (Pruss 1998, Wade 2003, Craun 2006, US EPA 2007, Boehm in press)

.

Pruss (1998) and Wade (2003) reviewed all the significant existing epidemiologic studies on the health effects from expo sure to recreational water. They found that most studies reported a dose

- related increase in swimming

- related illnesses associated with increases in recreational water indicator bacteria counts . The indicator organisms that reportedly associated best with the health outcomes were enterococci/fecal streptococci for marine and freshwater, and E. coli for freshwater. Of note, the majority of these studies were

### 7 cond ucted in the northern US and nor thern Europe , with few studies performed in sub/tropical marine

recreational waters and/or without known point sources

.

In a series of studies of randomly as signed bathers vs non

- bathers in the U nited K ingdom , Fleisher, et al. ( 1991, 1993, 1996, 1998) att empted to address some of the methodologic issues of prior studies. A summary of findings is presented in Table 1 . These results, coupled with the evidence that infectious microorganisms can occur in sub/tropical waters in the absence of specific contamin ation events and the di rth of information describing the impact of these illnesses (i.e., the severity of the illness or need for medical care), indicate d that the actual public health risk associated with bathing in sub/tropical waters containing microorg anisms, including those from EPA’s list of microbial indicators, is unknown.

Table 1 : Summary of results from the work of Fleisher et al. ( 1991, 1993, 1996, 1998) Number of Study Subjects Indicator (Concentration) Reporting Symptoms (Percentage) Stati stical Test Significance Fleisher et al (1993) Fecal streptococcus* GI Chi Square(trend)

278 Non

- bathers 7

- 14

78 <35 7

- 18

128 >35 20

- 24 P < 0.01

- 0.11 Fleisher et al (1996) Fecal streptococcus* (quartiles) Acute febrile Respiratory

illness Chi Square (trend)

665 Non

- bathers 3

124 0

- 14 4

138 15

- 27 5.8

125 28*50 1.6

158 51

- 158 7.6 P<0.04

Fecal Coliforms* Ear ailments

636 Non

- bathers 2.8

120 0

- 40 6.7

113 41

- 79 6.2

152 80

- 133 5.9

134 134

- 661 14.2 P < 0.001

Fleishe r et al (1998) Not specified GI illness Attributable Proportion

605 Non

- bathers 9.7

546 Bathers 14.8 34%

Acute Febrile Resp. Illness

665 Non

- bathers 3.0

546 Bathers 5.0 40%

Ear Ailments

636 Non

- bathers 2.8

525 Bathers 8.2 66%

Eye Ailments

658 Non

- bathers 2.1

538 Bathers 4.5 53% *Number of organisms per 100 ml

Finally, as noted above, beaches may also be impacted by non

- point sources. These are sources that can include storm water runoff, animal inputs, and/or human bather shedding (Gerba 2000, Elmir 2007, US EPA

### 8 2007, Boehm in press). In non

- point source beaches, the limited number of epidemiologic studies conducted

to date have not indicate d an assoc iation between enterococci (and other microbial indicators) and il lness

, even though some of these studies did find an association between bathing exposure and health risk ( Haile 1999, Fleming 2004, Colford 2007). Therefore, the association between illness and a specific microbial

indicator has not been well establish ed at beaches dominated by non

- point sources.

P RELIMINARY D ATA

The researchers (Solo Gabriele 2002, Fleming 2004, Shibata 2004, Elmir 2007, Abdelzaher 2008, Wright 2008, Solo

- Gabriele 2008) have conducted a series of studies to characterize the Study Si te, including extensive environmental monitoring, source tracking, experimental simulation, and small epidemiologic studies (including bather shedding studies).

The researchers conducted a pilot epidemiological and water quality study at two sub/tropica l public beaches, including the current Study Site, located in the southern part of Biscayne Bay, Miami (FL) next to both the University of Miami Rosenstiel School and the NOAA Southeast Center. This very small pilot study was funded by the NIEHS Marine an d Fresh Water Biomedical Sciences Center and the Miami Dade Dept of Health. The Study Site for original pilot and for the current Pilot Epidemiologic Study is approximately one mile long, relatively shallow, and characterized by poor water circulation. T he beach has a history of poor water quality: during 2000, the beach exceeded the EPA Poor Water Quality Guideline (PWQG) for enterococci 29.2% of the time. The results showed that there was no significant relationship between density of microbes and heal th effects although the sample size was very small (208 subjects) ; the water quality at comparison beach was better than the Study Site regardless of the season (wet vs. dry); there was no fecal pollution point source identified in the sanitary survey; int ensive spatial water quality monitoring indicated that the shoreline at the

### 9 Study Site appeared to be the source of microbes; and the concentrations of indicator microbes were usually lower during low tide as compared to high tide. The detection of those indicators at the shoreline without a known point source of fecal pollution again raised issues concerning the suitability of those indicators for measuring the recreational marine water quality in sub/tropical climates.

Over the past 4.5 years of NSF N IEHS Oceans and Human Health Center and collaborative NOAA and NSF funding, the Recreational Microbes Research Group has made significant progress. The group developed and implemented several sediment and water sampling and testing efforts within the Study Site. The overall results suggest that the inter

- tidal zone (sand that is periodically wet and d ry between low and high tide) may be the

source of indicator microbes at this beach. The release of indicators from this source is a function of tidal height , antecedent rainfall, wind direction and intensity, and other factors which influence microbial transport from the sediments. The sampling efforts supporting these observations have included: 1) intensive sediment sampling effort, 2) hourly sampling cont inuously for 48 hours, 3) sampling along transects at set tidal heights, 4) direct sampling of runoff, and 5) evaluation of the microbial contribution from runoff, human swimming and animal feces (Elmir 2006; Elmir 2007). Last summer, the research team co nducted a baseline assessment of the Study Site for the presence of pathogens with funding from the Florida Dept of Health . The results from the 4 sampling efforts showed that pathogens were detected when indicator microbes were elevated. Elevated

indica tor levels and pathogens were noted during a high tide sampling event characterized by low solar radiation. As a result of these prior activities at the Study Site, many important logistical, administrative and training activities were completed including : developing and implementing safety, sampling training and analyses protocols, meetings with the epidemiological, sediment and modeling groups, governmental agencies, and working with a variety of universities and public and private local laboratories. Th ose activities were essential for the success of the Pilot Epidemiologic Study discussed below.

S TUDY M ETHODS

The objectives of this Pilot Epidemiologic Study were to begin to evaluate the relationship between microbe densities and reported symptoms in human subjects with random assignment to recreational marine water exposure and intensive individual microbial monitoring in subtropical recreational marine waters with no known point source.

**Epidemiologic Methods** The design of the Pilot Epidemiologic S tudy was based on information collected by the Investigators d uring past studies at the Study Site described above (Shiba ta 2004, Fleming 2004 , Elmir 2007, Wright 2008 ) and on the work of Fleisher et al (1991, 1993, 1996, 1998) (see Table 1 above). The in vestigators conducted a

pros pective randomized study with study participant exposure randomly assigned to either potentially

contaminated marine recreational water or beach

- only exposure.

Local adult residents (at least 18 years old) who regularly repor ted bathing in recreational marine wate r s were recruited to participate in the study. After recruitment (see below) , the participants were screened for basic demographics, current illnesses and symptoms, alternative risk factors, and a brief health history ; they were then given an appointment for the study beach day

. At the study beach day, participants read and signed an IRB approved Consent Form (by Florida Dept of Health and University of Miami Human Subjects Committees; see Appendix) and a photography release. Participants were again interviewed briefly about current illnesses and symptoms, food consumption, and beach exposure since the baseline survey

. Study participants were then randomized to “water exposure” or “no water exposure ” (ie. bather vs no n

- bather, respectively). Study participants were instructed to: 1) Spend 15 minutes in knee

- deep water in a demarcated study environmental sampling area and dunk their heads completely unde r water 3 times (“bather ”), or 2) Spend time on the beach

at a dema rcated study environmental sampling site away from the water (“ non

- bather ”). After participating in the beach day protocol, participants were given a $50 American Express gift card for their cooperation in the study. They were also given an appointment to complete a n extensive follow

- up questionnaire seven (7) day after the beach visit. After completing the follow

- up questionnaire, study participants were sent an additional $25 American Express gift card to compensate them for any costs associated with par ticipating in the study.

### 10 Of note, all study materials were available in Spanish and English (see Appendix), and many of the study staff were bilingual. The questionnaires (as well as the study design) were adapted from those provided by Dr David Kay an d Dr Jay Fleisher from the prospective randomized intervention studies in Europe (see Appendix)

. Adaptations included some language changes to “American” English

, as well as logistical changes due to lack

of resources (instead of follow up at 7 days and 3 weeks, due to resources and logistics, these follow ups were combined into a single questionnaire and event at 7 days post exposure).

**Study Populations** : Only local adult residents (not tourists) were recruited to participate in the study due to logistics of the 7 day follow up

, as well as the possibility that tourists might represent an unusually susceptible subpopulation . An individual me t the study inclusion criteria if: 1) at least 18 years old; 2) regularly bathed in recreational marine waters; 3) li ved in South Florida at least 6 months or more per year; 4) did not have any chronic gastrointestinal (GI), respiratory dise ase, skin, or eye disease; 5) was willing to be exposed to bathing waters at the Study Site; 6) was willing to be randomi zed; and 7) understood and spoke either English or Spanish.

**Disease Endpoint Criteria** : The disease endpoints of interest in this study were the self

- reported symptoms of gastrointestinal illness, acute febrile respiratory illness (as specified by ICD 9 461

- 466, 480 ), eye or ear infections, and skin infections/irritation within seven days of going to the beach. These health endp oints were based on information collected during past studies by the Investigators at the Study Site and on the work of Fleisher et al. (199 1, 1993, 1996, 1998) (see Table 1 ). Of note, in this Report, only the results for gastrointestinal symptoms are reported.

### 11 **Recruitment Methods** : Volunteers were recruited by a variety of methods: word of mouth, newspaper articles, flyers, emails, as well as on

- site. Team members staffed booths at Miami

- Dade Community College, University of Miami Coral Gables Campus, Florida International University, and the Study Site to actively recruit individuals at these locales and to answer questions from potential p articipants. During recruitment, OHH employees fluent in both English and Spanish were present in order to remove potential selection bias. Potential participants were explained the qualifications for becoming a participant, instruction on study activities (including obtaining informed consent, completion of health data questionnaires, as well as the beach day activities), and contact information for registration purposes; these materials were also available in Spanish and English. Interested participants w ere allowed to select one day of the 15 possible on which to participate; subjects were only allowed to participate

once.

**Pre**

**- exposure Interview**  : Prior to arriving at the beach, participants verbally consented to complete a baseline health questionnaire document over the phone in Spanish and English. This was completed no more than 7 days prior to the sample collection day. Participants who had registered to participate on a specific sample collection day were telephoned each day during the week leading u p to the event in attempt to complete the baseline health questionnaire. An individual who had not completed the questionnaire prior to the sample collection day was not allowed to participate in the beach activities.

**Beach Study Day Data Collection** : Th e epidemiologic study data collection activities were conducted over a period of 15 days in coordination with the environmental researc h group beginning on December 15 , 2007 and ending on June 2 1, 2008. Each sampling day, 56

- 125 participants were randoml y assigned to spend a period

of 15 minutes in demarcated swim zones or 15 minutes in a demarcated non

- exposure zone(approximately 671 people/group overall). Overall study participation took between 30 minutes to 1.5 hours depending on the volume of partic ipants

, and was performed between 8 am and noon on a range of week and weekend days. On the actual study beach day, an IRB

- approved Consent Form was explained and signed, and then a second

pre

- exposure day interview was administered to each study participa nt. This interview was administered by a trained interviewer, focusing on any non

- water

- related behavior that could either cause or confound any association found between bathing and the outcome illnesses under study, any current illnesses, recent food

co nsumption, as well as any recent exposure to bathing waters. Participants reporting significant illness on the study day were excluded from the study.

Study subjects were randomly assigned to either a bather group or a non

- bather group. Individual bathe rs were then assigned to the bathing station where staff members supervised the exposure activity of the bather, including the time, location, unusual activities, and duration each individual bather spent in the water. Bathers were required to spend 15 m inutes in the water at knee

- deep water, and to immerse their head three times completely under water. Using ropes, a 20

- meter stretch of beach was subdivided into 5 meter intervals forming six bathing

exposure zones

. Exposure of any individual bather was therefore restricted to a single 5

- meter

- wide swim zone. Each subject was instructed on the technique to take their own water sample near the surface during their head immersions as well as provided with an appropriate water sampling container. Staff mem bers instructed participants to thoroughly rinse the collection container before filling completely with the marine water. Staff members observed the subjects at all times to insure that the subjects performed the water sampling appropriately

as well as fo llowed the 3 head immersion protocol and remained only 15 minutes in the water . When the subjects left the water, they gave their individual water samples to the Study Staff for microbial analysis processing (as described below).

No bather was allowed to enter the water more than once during the actual study exposure . The non

- bather group was restricted to a roped

- off area distant from the water and sand . Staff members were assigned to ensure

that non

- bathers did not enter the water. For logistical a nd safety reasons, the ratio of staff members to study subjects was approximately 1:9.

After all participants had completed their 15 minutes of “exposure,” they were thanked, given a hard copy of their Consent Form, an appoint ment for a telephone follow u p interview in 7 days, and a American Express Card Gift Certificate for $50.

### 12 **Follow up interview** : This short telephone follow

- up interview was used to assess the occurrence of illnesses (including questions about symptoms occurring since the beach visit, whether they sought medical care for their symptoms, and whether the symptoms caused them to miss work or school), as well as alternative risk factors for the study outcomes (including subsequent marine water exposures and food consumption) and subsequent beach visits. If participants were not able to be reached during the initial follow

- up call time, they were called back subsequent times between seven (7) and twenty

- one (21) days following the beach exposure. After completing the follow

- up questionnaire, study participants were sent an additional $25 American Express gift card through US Mail to compensate them for any costs associated with participating in the study.

**Environmental Sampling** The Study Site, (described above in Preliminary Data), has be en well characterized in terms of the microbial content of water and sediments including an identification of the sources of microbes to water and sediments. This characterization has been facilitated through a hydrodynamics model developed from a prior p ilot study and the ongoing research of the NSF NIEHS Oceans and Human Health Center at the University of Miami, as well as NOAA, Miami Dade County Public Health Department and CDC. Of note, the environmental monitoring and assessment are ongoing and funded by the NSF NIEHS Oceans and Human Health Center Recreational

Microbes Rese arch Project and collaborators

. The environmental assessment described below focus ed on the analysis of several microbes in water including pathogenic viruses, bacteria, and protozo a and the traditional

indicator microbes. Funds were provided by the FL DOH and CDC with the NSF NIEHS Oceans and

Human Health Recreational Microbes Research Project , NSF SGER and NSF REU , NOAA, and many collaborators for microbial analyses described belo w.

**Water Quality Monitoring at Study Site** : While each study participant was in the water, each subject collected their own environmental water sample from the demarcated study sites as described above. All water samples were placed in a cooler containi ng ice packs and transported back to the Microbe Laboratory at the University of

Miami Rosentiel School of Marine and Atmospheric Sciences (RSMAS) (<5 minutes by car and 1⁄4 mile) for processing.

Three different sets of samples were collected: 1) individua l; 2) composite; and

, 3) special samples . All samples were collected in the morning, approximately from 8 am to 12 pm on each sampling day. Individual water samples were collected at knee depth by epidemiological study participants using 5

- L sterilized p lastic containers after 15 minutes of bathing. Sampling points from the shoreline ranged from 10 to 40 m depending on tidal stage corresponding with the knee height. From 30 to 61 individual samples were collected per sampling day. Daily composite samp les were prepared by pooling 1 L of each individual sample collected on the day into a 50

- L sterilized carboy. For daily special samples, 10 liters of marine water were collected every

15 to 20 minutes from the time the first bather entered the water and pooled into two 250

- L sterilized plastic drums. In order to minimize the impacts from epidemiological study participants, the sampling point for the

“ special ” samples was approximately 5 to 10 m away from bathers.

Individual water samples were place in a cooler containing icepacks and after the collection of a batch of six, samples were transported to the microbial analysis laboratories at the University of Miami Rosentiel School (RSMAS) and National Oceanographic Atmospheric Administration Atlantic Oce anographic and Meteorological laboratory (NOAA/AOML), which are located in within a few minutes from the Study S ite (<5 minutes) for processing. The composite and special samples were kept outside but in the shade under tents during individual sampling, a nd then delivered to the RSMAS Lab shortly after the end of sampling efforts (around noon to 1 pm)

.

**Other Environmental Measurements** : Water temperature, pH, and salinity were measured for individual, composite, and special samples in the field using a Y SI probe (600R series sonde,YSI Inc., Yellow Springs, OH). Turbidity was also measured for these samples using a HACH turbidimeter. Additionally, other environmental conditions (e.g. tidal stage, rainfall, wind, and solar radiation) were obtained from Na tional Ocean and Atmospheric Administration (NOAA) and the University of Miami, RSMAS (NSF NIEHS OHH Center Remote Sensing Facilities Core).

### 13 Investigators also surveyed the bather density and animal populations in the study area during participant

expos ure, as well as evaluated data from a 24 hour 365 day per year digital camera focused on the Study Site and installed by the NSF NIEHS OHH Center Recreational Microbes Research Project with permission of Miami Dade County and assistance from the Miami Dade County Dept of Health and Miami SeaAquarium (Wright 2008). These data are being enumerated and evaluated by Dr John Wang and his research group as part of the ongoing development of an oceanographic fate and transport model for the Study Site.

**Microbial Assays**  : Individual samples were assayed for enterococci and Staphylococcus aureus . Daily composite samples made from individual samples collected on each day were assayed for multiple fecal indicator microbes (enterococci, C. perfringens , fecal coliform, E. coli , Bacteroides) waterborne pathogens (enterovirus, Cryptosporidium , and Giardia ), and S. aureus as a skin infection agent. In addition to these organisms, daily special samples were also assayed for enteric viruses, Cryptosporidium , and Giardia

.

**Fecal indicator microbes**

Enterococci : Enterococci, the microbial indicator organism currently recommended by the US EPA for marine waters, was enumerated using a traditional membrane filter method as recommended by the US EPA (USEPA 1997, 2000) an d a chromogenic substrate method as proposed by IDEXX known by the tradename

Enterolert TM (Westbrook, Maine) (APHA 1995). The membrane filter method involved placing the membrane filter containing the bacteria on a selective medium (mEI agar) and incubatin g the sample at 41°C for 24 hours. Colonies with a blue halo were then counted as enterococci. The chromogenic substrate method in simple

### 14 term utilizes enzymes specific to particular microbial groups. These enzymes were attached to dyes which

were then r eleased when the target microbe was present in the sample. Enumeration of the microbe population was based upon the use of a tray (Quanti

- Tray/2000) which separates the sample into a set of test

wells. The number of test wells that showed the characterist ic fluorescence under UV light were then counted and used in conjunction with a standardized table to provide the concentration in terms of the most probable number (MPN).

C. perfringens : There is scientific evidence that indicates that C. perfringens b ehave similar to enteric viruses

in marine and fresh waters

, and that this bacteria does not multiply outside the human gastro

- intestinal tract under aerobic conditions (Fujioka 1985; Fujioka 1997). Therefore, using C. perfringens as part of a monitoring

p rogram for the microbiological recreational water quality can be a valuable alternative, particularly in sub/tropical marine environments, where the regrowth of the other indicator microbes is suspected to occur.

The state of Hawaii has elected to use C. p erfringens in their water quality monitoring, and the USEPA has recently approved a membrane filtration method to detect and enumerate C. perfringens spores in water and

sediments. C. perfringens was analyzed using the membrane filter method (USEPA 1995) r equiring the use of mCP agar. Samples were incubated anaerobically at 44.5± 0.2°C for 24 hours. Plates were then exposed to ammonium hydroxide fumes after the incubation and dark pink to magenta colonies were counted as C. perfringens

.

Fecal coliform s : Fecal coliform s were o riginally recommended by the US EPA for recreational water monitoring (U.S.EPA 1976)

, and still used by the Florida Department of Health (FL DOH) and Florida Department of Environmental Protection (FL DEP). Enumeration of fecal colif orm s was based upon the MF method (APHA 1995; Method 9222 D). Filters were placed on modified mFC agar (Becton Dickinson, Sparks, MD) and were

incubated a t 44.5 ± 0.5oC for 24 hours; colonies that were various shades of blue were counted as fecal

coliform s

.

E. coli : The use of E. coli is recommended for fresh water monitoring by the US EPA ( US EPA 1986). The MF method was used for enumerating using modified mTEC agar ( US EPA 2002, Method 1603). Samples were

incubated at 35 ± 0.5°C for 2 hours

, and then i ncubated at 44.5 ± 0.2°C for 22 hours. Target red or magenta colonies were counted and reported as E. coli

.

Bacteroides : Bacteroides are found in the intestine of humans and other warm blood animals ; thus

, detection of bacteroides in water implies that the water has been contaminated with human or other animal feces. For B.

thetaiotaomicron analysis, 400 mL of water from each sample was filtered through a 0.20 μm polycarbonate filter (Millipore Corp., Billerica, MA). The polycarbonate filter was placed into a 15mL conical tube. The tube was vortexed for 2 minutes and the filter removed. Conical tubes were first concentrated by centrifugation at 2052 g for 15 minutes at 4oC. The supernatant was discarded, except for 200 μl, which was used to resuspend t he pellet. DNA was extracted with the POWERSOIL DNA Extraction Kit (MOBIO Laboratories, INC.). The extracted DNA was analyzed for presence of B. thetaiotaomicron by PCR (Eppendorf Master Mix 2.5, Brinkman Instruments, Inc, Westbury, NY) with primers (B.th etaF and B.thetaR).

**Pathogenic microorganisms** Due to presumption of low concentrations of pathogens in the recreational marine water from nonpoint sources of fecal contamination, daily composite and special samples were concentrated by two different meth ods. The first method of sample processing used traditional large volume filtrations. EPA method 1623 ( Cryptosporidium and Giardia in water by filtration/IMS/FA) for protozoan pathogens and EPA’s Manual for Virology (Chapter 14, Concentration and Processi ng of Waterborne Viruses by Positive Charge 1 MDS Cartridge Filters and Organic Flocculation) were followed with the exception of: a) type of filter used, as positively charged filters are adequate for freshwater only; b) the addition of acid and salt cons istent with the needs of a negatively charged filter; and. c) the option to add acid and salt using an equilibration basin or fluid proportioner system. Negatively charged filters were utilized in accordance with those specified through

Standard Methods fo r the Analysis of Water and Wastewater , Section 9510 C

– Virus Concentration from Large Sample Volumes by Adsorption to and Elution from Microporous Filters, 21st Edition, 2005. The preparation of feed solutions for electronegative filters followed method s as outlined in Standard Methods

,

### 15 Section 9510 C. The elution and virus culture procedure followed EPA’s ICR Microbial Laboratory Manual

, Section VIII

– Virus Monitoring Protocol for the ICR, 1996. The method of acid and salt addition used an equilibrati on basin as described in EPA Method's 5 and 6 from the Manual for Virology ). The second method of sample processing, called the bilayer method, used 10 L of composite sample (Abdelzaher 2008). The top and bottom filters were eluted separately for protozoa n ( Cryptosporidium and Giardia ) and viruses (norovirus, enterovirus, and Hepatitis A virus).

Water concentration for viruses and protozoa was conducted at the NSF NIEHS OHH Lab, RSMAS, University of Miami. Once concentrated onto filters, these samples were sent to universities or government laboratories for elution and analysis.

Pathogenic Viruses : The presence of enteric viruses in coastal waters pose a public health risk since these viruses are typically transmitted via the fecal

- oral route, and be cause for many viruses, only a small number of virus particles can initiate an infection (Haas et al. 1999) . Because both the Florida Keys and the Tampa Bay region have been investigated for the presence of viruses in coastal waters and the presence of en teric viruses

has been confirmed (Griffin 2003) , it is likely that investigatin g the Miami region of Florida would also reveal the presence of viral pathogens in the coastal environment

. After concentration of the special samples using

Filterite filters (with pH and salt adjustment), the samples were sent to BCS laboratories in Gain es ville, FL for elution and analysis for enteric virus using traditional virus culture methods. Concentrated composite samples were assayed for enterovirus based on qPCR

.

Pathogenic Protozoa : For the special samples, the filters were eluted, the pellet was concentrated, and the Cryptosporidium and Giardi a were purified using immuno

- magnetic separation. The purified pellet was stained with a fluorescent antibody and the nu mber s of oocysts/cysts were counted using a microscope. Protozoan in the composite samples were assayed based on qPCR.

Staphylococcus aureus : S. aureus has been commonly associated with skin infections in marine waters (Charoenca 1993, Charoenca 19 95). S. aureus was assayed based culture method using Baird

- Parker agar base with Egg Yolk Tellurite Enrichment followed by a series of confirmation tests using Mannitol Salt agar, Latex agglutination, and PCR. Furthermore, the presence of Methicillin

- re sistant S. aureus ( MRSA) was

identified base on PCR.

**Epidemiologic Study Sample Size Calculations** : The sample size for the epidemiologic study was calculated based on information collected during past studies by the Investigators at the Study Site and on the work of Fleisher et al., ( 1991, 1993, 1996, 1998) (see Table I). Using GI symptoms as reported by Fleisher et al (1993)

and assuming 95% confidence and 80% power, approximately 1300 study participants (650 bathers and 650

non

- bathers) were needed to s how an increase in reported GI symptoms of 5% in bathers vs. Non

- bathers.

**Randomization, Epidemiologic Study Database & Statistical Analysis**  : Block randomization was used to maximize the probability of achieving equal number of study participants in th e 2 groups under study. Block size consisted of a random ordering of blocks of two, four and six study participants per individual block. It should be noted that the random ordering of block size eliminates any chance of clinical personnel discovering th e blocking
pattern. Block sizes of two, four and six were chosen to maximize the probability of equal numbers of participants in each study group. All participants were not told their randomized status until completing the exposure day

interview on the b each.

The Study Database was created in ACCESS. The data were analyzed using the statistical software package,

SAS

, for the purposes of the epidemiologic analyses. All univariate analyses were performed using the SAS package of statistical programs. Si mple s tratified analyses were conducted using EPINFO, whi le all logistic modeling was computed using SAS

.

Initial analyses evaluate d the effects of randomization for all demographic variables to insure equal distribution of demographic risk factors among exposed (ie. bathers) and unexposed (ie. non

- bather s) populations. The basic analyses were geared to answer two questions: (1) "Is there an excess of the illnesses under study among

### 16 bathers vs. non

- bathers?"; and (2) "Is there a dose

- response relationship between indicator organism density and the incidence of the outcome illnesses studied among bathers?"

1) Bather vs. Non

- Bather Comparisons : Success of randomization was assessed by comparing the distribution of

non

- water

- related risk factors for the il lness under study among bathers vs. non

- bathers using univariate chi

square analysis. Where expected cell frequencies were less than 5.0, Fishers Exact Test (2

- tailed) was used in

lieu of the Pearson X 2

The results of the environmental monitoring are presented first

, followed by the descriptive results of the epidemiologic study and by the results of the hypotheses which combine both the epidemiologic and

environmental data . Of note, since data collection was completed at the end of June 2008, there continue to

be ongoing microbiologic and statistical analyses. Thus, limited data results are presented below with the

expe ctations that addi tional results will be published subsequently in peer reviewed publications. In particular, only gastrointestinal symptoms are reported for the following reasons: a) these were the most numerous symptoms reported and thus yield the most statistically robust analyses; b) these are the symptoms most commonly associated with exposure to recreational marine waters in the literature ; and c) these are the symptoms upon which the EPA regulations are based for the mo nitoring of these waters using e nterococcus as statistic. Assuming randomization was successful, the crude rates of illness for bather s and

non

- bathers were adjusted for any of these extraneous ri sk factors. If randomization was not successful for any

non

- water

- related risk factor, the effect of this failure was assessed for any confounding of the assoc iation found between illnesses among non

- bathers vs. bathers.

A se cond chi

- square analysis was conducted for each of the illnesses under study to assess at what indicator organism density rates of illness among bathers we re significantly greater than ra tes of illness among non

- bathers. In the context of public health planning, specifically the setting of recreational water qualit y criteria, this analysis provide d insight into possible thresholds of effect (i.e., an indicator organism density below which bathers experience no excess illness relative to non

- bathers). It must be emphasized that this analysis wa s not meant to definitively define such a threshold, but rather to generate hypotheses regarding the possible existence of such

thresholds to be tes ted by future epidemiological/statistical research, particularly for sub/tropical locations without known point sources.

2) Analyses Restricted To Bathers Only : In order to assess the relationship between indicator organism density

and each illness under study, while identifying and controlling for non

- water

- related risk factors fo r such illness, analyses were restricted to bathers only. Exposure (i.e., indicat or organism density) was related to each bather using the time an individual exited the water, t he swim zone used, and the indicator organism density recorded. .

Chi

- square analyses were then used to identify any statistically significant trends among the observed incidence of a particular outcome illness among non

- bathers vs. bathers at increasi ng levels of indicator org anism exposure.

Bathers were divided into different distributions of indicator organism exposure (e.g quartiles) , and trend s then assessed for each indicator organism assayed. These series of analyses included the rates of illne ss observed

among non

- bathers. When a particular outcome illness did not show a statistically significant trend for any of the indicator organisms assayed, rates of illness among bathers in the highest quartile of indicator organism exposure were compared to rates of illness among bathers in the lowest quartile using chi

- square analysis.

Multiple logi stic regression modeling was used to evaluate relationships between indicator organism density and illness among bathers while controlling for extraneous non

- water

- related risk factors identified via a prior univariate chi square analysis of postulated non

- water

- related risk factors or confounders among ill vs. non

- ill bathers. Initial criteria for entering non

- water

- related risk factors or possible confounder s into the l ogistic regression model were at p < 0.15, although retention in the model was at p<0.05. A backward step

- w ise elimination strategy was employed in logistic regression modeling used to control for non

- water

- related risk factors or confounder s for the illness under analysis. For convenience, based on the workings of the statistical package used for logistic regression modeling, a for ward step

- wise procedure was used to assess the magnitude of any confounding of any observed indicator organism exposure

- disease outcome relationships by non

- water

- related risk factors.

R ESULTS

### 17 the recommended indicator organism . The possible association between gastrointestinal symptoms and the

ind icator organism, enterococcus, wa s evaluated using enterococcus as analyzed by the membrane filtration method. In addition, althoug h some data are presented for the pathogen analyses, no statistical analyses were performed of these particular data . Again, i n the future, the Research Group plans to expand on the results of this Report by publishing in the peer

- reviewed scientific lite rature.

**Overview of Environmental Results**

Microbial Concentrations

The following microbial analyses results are provisional and pending final review; subsequent papers in the peer reviewed literature will contain results which will supercede the microbi al results outlined below.

Individual Samples : A total of 668 water samples collected by individual epidemiologic participants were assayed for enterococci and Staphylococcus aureus over 15 individual study days from December 2007 through June 2008 ( Tabl e 2 ). Although some of these waters samples were not used in the epidemiologic analyses due to loss to follow up (see above), the data presented below are for all 668 samples.

Table 2 : Summary of individual enterococci and S. aureus levels for each moni toring day.

Date N Enterococci CFU/100ml (MF) Enterococci MPN/100ml (CS) S. aureus CFU/100ml

Min Max Ave GM Min Max Ave GM Min Max Ave GM

12/15/07 30 1 83 6±15 2 10 120 26±29 17 1 60 6±12 2

01/12/08 34 1 123 27±34 11 10 201 44±48 27 1 760 40±135 3

0 1/26/08 42 1 1580 65±242 12 5 152 53±39 38 1 100 12±22 3

02/16/08 48 1 559 27±85 4 5 316 39±51 23 1 400 30±84 3

02/23/08 53 1 271 28±41 15 5 137 28±25 20 1 220 17±41 3

03/08/08 38 19 3320 350±817 98 20 2844 310±710 88 1 200 11±35 2

03/22/08 38 2 757 14 9±184 58 5 644 123±133 67 1 20 3±4 2

03/29/08 55 1 219 20±34 8 5 73 13±13 9 1 532 14±72 2

04/12/08 49 1 159 33±39 13 5 120 19±22 12 1 440 18±66 3

05/10/08 53 1 1760 66±244 13 5 163 43±37 28 1 200 10±33 2

05/18/08 52 20 618 100±91 81 5 888 108±122 81 1 2 1±0.2 1

06/01/08 61 1 820 75±114 34 5 311 63±65 35 1 240 22±44 5

06/07/08 53 1 1140 34±159 5 5 74 23±19 16 1 40 4±7 2

06/13/08 31 1 621 66±146 10 5 453 47±87 21 1 780 41±141 5

06/21/08 30 10 849 73±152 40 5 693 94±122 63 1 560 40±104 7

Ave 44 4 859 75±160 27 7 479 69±101 36 1 304 18± 53 3
N is the number of individual samples collected on each sampling date; Min and Max are lowest and highest microbial levels on the day; Ave is arithmetic mean with standard deviation; GM is the geometric mean.

Enter ococci were measured based on the membrane filter (MF) and chromogenic substrate (CS) methods

Enterococci : The concentration of enterococci in individual samples ranged from the detection limit of 2 to 3320 CFU/100mL with an approximate average of 7 5 ( st andard deviation, ± 160 ) CFU/100mL based on the MF method. Those samples with below detection limit values were set at half the detection limit value (1 CFU/100mL) for the statistical analyses. The enterococci concentrations based on the CS method ranged from below 10 to 2840 MPN/100mL with an approximate average of 6 9 ±1 01 MPN/100mL. Overall, enterococci levels based on the MF and CS (n = 668) were statistically not different. The two methods were strongly correlated (r = 0.87) for higher values (when MF values were larger than 35 CFU/100mL). The correlation between the methods was weak (r = 0.19) for MF values equal to or below 35 CFU/100mL.

Staphylococcus aureus : The concentrations of S. aureus ranged from below 2 to 780 CFU/100mL with an approximat e average of 1 8 ± 53 CFU/100mL based on the cultured method followed by PCR confirmation.

Methicillin Resistant S. aureus (MRSA) was detected from 1% (7/668) of individual samples. The

### 18 concentrations of MRSA ranged from below 2 to 68 with an approximate av erage of 1±3 CFU/100mL. The

levels of S. aureus and enterococci were not correlated (r < 0.01).

Daily Samples : Results of microbial assays from 15 composite and 15 special samples were summarized in

the Tables 3 and 4

.

Table 3 : Multiple fecal indic ator microbes in daily “composite” samples

Date Enterococci Clostridium perfringens Fecal

coliform E. coli Bacteroides

thetaiotaomicron

MF CS MF MF MF PCR

CFU

/100ml MPN

/100ml CFU

/100ml CFU

/100ml CFU

/100ml per 400ml

12/15/07 1 55 1 3 1

- 01/12/0 8 15 52 <0.7 104 1 + 01/26/08 4 61 7 27 31

- 02/16/08 15 18 <0.7 10 3 + 02/23/08 13 13 1295 20 7 + 03/08/08 99 69 1653 196 83

- 03/22/08 109 100 402 61 27

- 03/29/08 1 23 13 66 3 + 04/12/08 3 13 4 1 1

- 05/10/08 29 124 5 5 3

- 05/18/08 50 43 34 9 23

- 06/01/08 13 15 24 127 25

- 06/07/08 <0.7 11 6 7 <0.7

- 06/13/08 14 48 159 159 30

- 06/21/08 1 68 16 147 17

- Ave 25 47 241 63 17
MF and CS stand for membrane filter and chromogenic substrate methods, respectively;

Ave is the arithmetic mean of 15 daily samples; One half the detection limit was used for samples measuring below detection limit values.

**Fecal Indicators**

Enterococci : T he concentrations of enterococci in daily composite samples ranged from below the detection

limit of 2 to 109 CF U/100mL with an approximate average of 2 5 ±36 CFU/100mL based on the MF method. The enterococci concentrations based on the CS method ranged from 11 to 124 MPN/100mL with an average of 47±34 MPN/100mL. Enterococci levels based on the CS method showed slig htly higher results than the

MF

, but statistically not different (p = 0.08).

C. perfringens : The concentrations of fecal coliform in the composite samples ranged from below 2 to 1650

CFU/100mL

, with an average of 2 41 ±547 CFU/100mL based on the MF metho d.

Fecal coliform s : The concentrations of fecal coliform s in the composite samples ranged from 2 to 196

CFU/100mL

, with an average of 63±67 CFU/100mL based on the MF method.

E. coli : The concentrations of E. coli in the composite samples ranged fro m below 0.7 to 83 CFU/100mL with an approximate average of 1 7 ±22 CFU /100mL based on the MF method.

Bacteroides thetaiotaomicron was detected in four composite samples (per 400 mL) base d on PCR.

**GI Pathogens**

Enteric virus: The concentration of enteric v irus in the daily “special” samples ranged from below detection limit of 1 to 2.2 MPN/100L. The concentrations of Pan Specific Enterovirus in the daily “composite” samples were below detection limits (0.5

– 1.1/100mL) for all daily composite samples.

### 19 C ryptosporidium: Cryptosporidium was detected from only one daily “special” sample (1.6 oocysts/100L or 0.002 oocysts/100mL), while other samples were below the detection limit of 1 oocyst/100 L or 0.001 oocyst/100mL. The concentrations of Cryptosporidiu m in the daily “composite” samples were all below detection limits ranging from 0.1 to 0.3 oocysts/100mL.

Giardia: Giardia were detected in two “special” samples (1.1 and 2.1 cysts/100L or 0.001 and 0.002 cystes/100mL), whereas the remaining samples we re below the detection limit of 1 cyst/100L or 0.001 cyste/100mL. The concentrations of Giardia in the daily “composite” samples were all below detection limits, ranging from 0.1 to 0.3 cysts/100mL.

Table 4 : Pathogens in daily samples (composite and * special)

Date *Enteric

virus Entero

virus Cryptosporidium Giardia Staphylococc

us

aureus
*Culture qPCR *IMS

- FA qPCR *IMS

- FA qPCR MF

/100ml /100ml /100ml /100ml /100ml /100ml /100ml

12/15/07 0.0016 <0.5 0.0016 <0.1 0.0011 <0.1 8

01/12/08 <0.0005 <0.5 <0.0001 <0.1 <0.0001 <0.1 9

01/26/08 <0.0005 <0.5 <0.0001 <0.1 <0.0001 <0.1 24

02/16/08 <0.0005 <0.5 <0.0001 <0.1 <0.0001 <0.1 6

02/23/08 <0.0005 <0.5 <0.0001 <0.1 <0.0001 <0.1 2

03/08/08 <0.0006 <1.1 <0.0001 <0.3 <0.0001 <0.3 <0.7

03/22/08 <0.0006 <0 .7 <0.0001 <0.2 0.0021 <0.2 5

03/29/08 <0.0005 <0.5 <0.0001 <0.1 <0.0001 <0.1 <0.7

04/12/08 <0.0005 <0.5 <0.0001 <0.1 <0.0001 <0.1 11

05/10/08 0.0022 <0.5 0.0009 <0.1 0.0023 <0.1 <0.7

05/18/08 0.0005 <0.6 <0.0001 <0.1 <0.0001 <0.1 <0.7

06/01/08 <0.000 6 <0.7 0.0010 <0.2 <0.0001 <0.2 14

06/07/08 <0.0005 <0.5 <0.0001 <0.1 0.0015 <0.1 12

06/13/08 0.0005 <0.5 <0.0001 <0.1 <0.0001 <0.1 2

06/21/08 0.0005 <0.5 0.0005 <0.1 0.0010 <0.1 13

IMS

- FA is i mmunomagnetic separation and immunofluorescent assay

**Skin Pathogen**  **s** S. aureus : The concentrations of S. aureus ranged from less than 2 to 24 CFU/100mL with an approximate average of 10±6 CFU/100mL.

Physical, Chemical, and Hydro

- meteorologic Parameters

Time: The Study Site water quality was evaluated for 15 d ays from December 15, 2007 to June 21, 2008. The individual samples were collected between 7:44 to 11: 52 AM with an average time of 9:52 AM.

pH: The pH values of individual samples ranged from 6.05 to 8.82 with an average of 7.97± 0.30. The average

p H of daily individual (sample mean, μ = 7.95 ±0.26), composite (μ = 8.02±0.28), and special samples (μ = 7.94±0.35) were statistically the same.

Salinity: The salinity values of individual samples ranged from 32.12 to 38.58 ‰ with an average of

35.73±1. 18 ‰ . The average salinity of daily individual (μ = 35.65±1.32 ‰ ), composite (μ = 35.64±1.43 ‰ ), and special samples (μ = 35.77±1.28 ‰ ) were statistically the same.

Temperature: The temperatures of individual samples ranged from 20.6 to 31.3 oC, with an a verage of 26.0±2.3oC. The average temperatures of daily individual (μ = 25.3± 2.6oC), composite (μ = 26.4±3.0oC), and special samples (μ =25.8±3.0oC) were statistically the same.

### 20 Turbidity : The turbidity values ranged from 0.4 to 117 NTU

, with an aver age of 11.8±13.5 NTU for the individual samples. The average turbidities of daily individual (μ = 12.4±13.2 NTU), composite (μ = 14.5±14.7NTU), and special samples (μ = 12.1±9.1 NTU) were statistically the same.

Tide : The tidal stage ranged from

- 0.1 to 0.8 m with an average of 0.2±0.2 m during individual sampling.

Rainfall : Only 3 rainfall events were observed during monitoring efforts. Cumulative rainfalls in the previous six hours and during sampling were 12, 0.51, and 0.77 mm on March 8, May 18 , and June 1, 2008, respectively.

Wind : Wind direction ranged from 10 to 310o , with an average of 149±79o. T he wind speed ranged from 2.4 to

11.3 m/s

, with an average of 5.8±2.4 m/s during individual sampling.

Solar Radiation : The solar radiation r anged from

- 13.9 to 35.5 W/m 2

, with an average of

- 0.5±3.4 W/m 2 during field sampling.

**Epidemiologic**

Demographics of Study Population: There were 1341 subjects who participated in the 15 beach study days from December 2007 through June 2008 ; 38 (2.9 % were lost to follow up) resulting in a total cohort of 1303. After randomization, there were 652 bathers and 651 non

- bathers ( Table 5 ). Based on the se results ( as well

as data not shown ) , the randomization of subjects into bather and non

- bather exposure f or the purposes of the study was successful in terms of equal distribution of demographics.

T able 5. Demographics of Study Population

**H1 : People exposed to recreational waters contaminated with infectious microorganisms will be**  **significantly more likely to report symptoms than unexposed similar people who go to the**  **beach but do not go into the water.**

**Gastrointestinal (GI) Symptoms** For the purposes of these analyses, of the 1341 participants enrolled in the study, 38 (2.91%) were lost to

follow

- up. Of the remaining 1303 stu dy participants, 64 (4.8%) , reported having a chronic condition that predisposed to symptoms of gastroenteritis (e.g., colitis, irritable bowel syndrome, etc.), or reported symptoms of gas troenteritis on the exposure day , and were excluded from the study a nalysis for gastrointestinal
symptoms . Therefore the final sample size for the gastrointestinal symptom analyses was 1239.

Prior to analysis, gastrointestinal symptoms were categorized as either: all cases of vomiting or diarrhea, or all reported cases of indigestion or nausea accompanied by a fever. “Diarrhea” was defined as having three or Variable All Participants Bather Non

- bather p

N (%) Bather Status 1303 (100) 652 (50.04) 651 (49.96) Mean Age (+/

- SE) 32.39 +/

- 12.97 22.98 +/

- 253.68 29.01+/

- 57.92 0.52 N (%) Female 751 (57.64) 370 (49.27) 381 (50.73) 0.54 N (%) Hispanic ethnicity 499 (38.30) 243 (48.70) 256 (51.30) 0.40 N (%) Occupation

- Office

- Catering/leisure

- Agriculture

- Student/School/Universi ty

- Factory

- Caring for Others

- Building/Construction

- Other 1299 (100) 295 (22.71) 14 (1.08) 10 (0.77) 572 (44.03) 8 (0.62) 62 (4.77) 37 (2.85) 301 (23.17) 141 (47.80) 9 (64.29) 5 (50) 285 (49.83) 7 (87.50) 26 (41.94) 23 (62.16) 154 (51.16) 154 (52.20) 5 (35.71) 5 (50) 287 (50.17) 1 (12.50) 36 (58.06) 14 (37.84) 147 (48.84) 0.12

SE= Standard Error

### 21 more runny stools within a 24

- hour period. This categorization of symptoms of gastroenteritis was, therefore, less likely to be affected by inter

- individual variation in perception of specific symptoms, and thus provided a

more reliable estimate of t he occurrence of gastrointestinal symptoms among study subjects. Furthermore, this categorization of symptoms was less likely to be subject to any recall bias among bather s or non

- bathers, given the nature of the symptom and the relatively short period between exposure and follow

- up. In addition, this categorization of symptoms was quite similar to those used other published large prospective study of

recreational waterbor ne illness

, and thus provide s the opportunity for comparison of results Fleisher et al. (1991, 1993, 1996, 1998).

Overall, a nalysis of the crude rates of gastrointestinal symptoms showed bathers to be 1.76 times more likely

to suffer gastrointestinal symp toms relative to Non

- bathers ( odds ratio=1.76; 95% Confidence interval (CI)=0.98

– 3.19; p=0.057). A variety of confounding factors were considered (only statistically significant comparisons shown), including: age, gender, number of people in household, med ications and illnesses in the weeks prior to the study exposure, the foods consumed from 3 days prior to throughout the 7 day follow up period including any “take out” food (bathers vs non

- bather s: 54.2% vs 45.8%; p=0.02) , illness in the household througho ut the 7 day follow up period (69.6% vs 34.4%; p=0.0002) , and additional bathing during the 7 day follow up period.

The crude rates shown above did not take into account the possible confounding effect due to non

- bathing

water

- related risk factors for g astro enteritis. Adjustment for possible confounders/extraneous risk factors ( Table 6 ) still demonstrated that Bathers were at the same elevated risk relative to non

- bathers (1.76 (0.94

- 3.30); p=0.07) . T hese analyses confirmed that the observed diffe rences in the overall rate of gastrointestinal
symptoms among bathers vs. non

- bathers were not confounded by the non

- water

- related risk factors evaluated in this study.

Table 6. Results of Unconditional Multiple Logistic Regression Analysis: Bathers vs Non

- bat her s Risk factor Chi

square P

value Odds Ratio (95% Confidence Interval) GI Symptoms 3.17 0.07 1.76 (0.94

- 3.30) Excessive tiredness 3 week prior to initial interview lasting <24 hours 7.21 0.007 4.65 (1.51

– 14.27)

Eat salad after Trial day 4.54 0.03 0.75 (0.57

– 0.98) Anybody else in household ill since exposure day 7.91 0.005 0.49 (0.30

- 0.81)

Eat Ice

- Cream since trial day 3.50 0.06 0.79 (0.62

- 1.01)

**H2: The organism densities sampled at the time of exposure in the marine waters wil**  **l be associated**  **with reported human symptoms**

**Gastrointestinal (GI) Symptoms & Enterococcus (MF)** The crude rates of GI symptoms are shown in Table 7 among non

- bathers compared to bathers at quartiles of

Enterococci exposure ( personal samples by the membrane filtration method). T here was a statistically significant trend between increasing Enterococci counts and increasing risk of gastrointestinal symptoms (p=0.04). Using Enterococci as the measure of exposure, there was a statistically significantly inc re ased risk of 2.89 times for gastrointestinal symptoms for Bathers compared to non

- bather s at highest quartile of exposure ( 2.89; 1.23

– 6.76 ; p=0.006 ).

The crude rates of GI symptoms among non

- bathers compared to bathers in increasing 20 unit intervals of Enterococci exposure are shown in Table 7 . There was a statistically significant trend between increasing Enterococci counts and increasing risk of GI symptoms (p=0.01). There was an increased risk of 2.47 times for GI symptoms for Bathers compared to non

- bathers at highest quartile of exposure (0.96

- 6.10; p=0.052), although not quite reaching statistical significance. Bathers exposed to greater than 40 Enterococci at higher risk relative to bathers exposed to less than 40 Enterococci /100 mL of individual sample. In addition, bathers exposed to less than 40 enterococci/100 ml had rates of illness similar to the non

- bathers.

### 22 The crude rates shown above did not take into account the possible confounding effect due to non

- bathing

water

- related risk factors f or gastro

- enteritis. Subsequent analyses, therefore, assessed the effect of enterococcus on the reported illness among bathers, to assess the effect of confounding by non

- water

- related risk factors for gastroenteritis, including diarrhea lasting 24 hours within 3 weeks of the study (Ill Bather vs non ill bather: 25.0% vs 75.0%; p=0.06), and taking any medicines in the 4 weeks prior to exposure (8.1% vs 91.9%; p=0.008).

Table 7. Crude Rates of gastrointestinal s ymptoms among non

- bathers vs. bathers by Ent erococcus levels in personal samples

Exposure Status N Rate/100 P value (Trend)

Enterococcus Quartiles

Non

- bather s 620 2.90 0.04 Bathers (0

- 4) 171 5.85 Bathers (5

- 20) 145 2.07 Bathers (21

- 61) 149 4.70 Bathers (61

- 3320) 99 4.14

Enterococcus cou nts by 20

Non

- bather s 620 2.90 0.01 Bathers (0

- 20) 306 4.25 Bathers (21

- 40) 102 2.94 Bathers (41

- 60) 53 7.55* Bathers (61

- 80) 40 7.50* Bathers (81+) 118 6.78* Enterococcus measurements by membrane filtration method; *p < 0.10

Unconditional mu ltiple logistic regression analysis was used to model the relationship between Enterococci exposures less than vs. greater than or equal to 40 CFU/100 mL and GI illness among bathers while controlling for non

- water

- related risk factors/confounders. Thus, bathers were at 2.05 greater risk of being ill when exposed to enterococcus levels greater than or equal to 40 CFU/100 mL than bathers exposed to enterococcus levels less than 40 CFU/100 mL (2.05 (0.97

- 4.30); p=0.059) ( Table 8 ).

Table 8 . Multiple logistic regression analyses among Ill vs Non Ill bathers and enterococcus exposure less than

40 CFU/100 mL vs. greater than 40 CFU/100 mL while controlling for possible non

- water related risk

fac t ors/confounders.

Risk factor Chi square P value Odds Ratio (95% Co nfidence Interval)

Enterococcus * 3.58 0.059 2.05 (0.97

– 4.30)

Diarrhea 3 or more times lasting 24 hours during the 3 weeks prior to exposure 4.21 0.04 0.17 (0.03

- 0.92)

Taking Tablets/ Medicines taken 4 weeks prior to exposure 5.91 0.01 0.39 (0.18

– 0.83)

*Enterococci modeled less than 40 vs. greater than or equal to 40 enterococci/100 ml.

D ISCUSSION

The objectives of this Pilot Epidemiologic Study were to begin to evaluate the relationship between microbe densities and reporte d symptoms in human subjects with random assignment to recreational marine water exposure and intensive individual microbial monitoring in subtropical recreational marine waters with no known point source. Our study results indicate d t hat there wa s an inc reased risk of reporting symptoms with bathing exposure at the non point source subtropical recreational marine wa ter. Furthermore, there appeared to be an

### 23 association between enterococcus (by membrane filtration method) levels and report of bather sympto ms at or

above 40 CFU/100 mL of enterococcus based on an individual sample

. The Study Site, which is a non

- point source of fecal contamination, showed high levels of indicator microbes intermittently during this study.
Nevertheless, these intermittent hig h levels of indicator microbes did not correlate on a one

- to

- one basis with the presenc e or absence of these pathogens; s ometimes pathogens were detected when enterococci levels

were elevated

, and sometimes pathogens were not detected during periods of ele vated enterococci (see

Table s 2

- 4 )

.

**Environmental Sampling** Water samples collected by individual epidemiologic study participants contain elevated levels of enterococci. The percentage of the total individual samples that exceeded the single sample limit of enterococci (104/100mL) as recommended in the US EPA guideline were 13% based on the MF and CS methods. This result was consisted with the monitoring data as collected by the Florida Department of Health (FL DOH 2008)

which also monitored the same Stu dy S ite during the previous year. During four days (March 8th, March 22nd, May 18th, and June 21st, 2008) during the 15

- day monitoring period, the geometric means of individuals on each sampling day exceeded 35/100mL (US EPA 1986) based on the MF and CS methods . Additionally, the geometric mean of individual samples on January 26th, 2008 also exceeded 35 /100 mL based on the CS

method . S. aureus were detected from most individual samples

, but there is no guideline for dermatitis illness.

S. aureus level s were neither correlated with enterococci nor environmental parameters . T he source of S.

aureus is believed to be the bathers themselves.

Viruses and protozoans were not detected from the composite samples unlike the special samples. This

resul t was not likely due to differences in the samples

, but rather to the different sample concentration methods used to process each type of sample. The special samples were processed based on the conventional large filtration method while the composite samp les were processed based on the alternative Bilayer method, which is applicable for small water sample volumes with high concentrations of mic robes. Since the water at the Study S ite contained very low levels of viruses and protozoan, they were not detect ed from the composite samples which were based on the analysis of smaller sample volumes.

**Limitations and Strengths**  (Pruss 1998, Wade 2003, Fleisher 2006, Craun 2006, EPA 2007, Boehm in press)

There are a number of limitations to this Pilot E pidemiologic study, in part due to limited resources. First, there was no objective measurement of symptoms or illness, but rather self report; ideally, objective ascertainment by healthcare providers at 7 and also if possible 21 days after exposure would be important. Of note, future

data analyses will be performed to evaluate the possibility that increased risk perception may lead to increased self report of disease by the participants (Fleisher 2006).

The lack of children in this study was also a li mitation since children have been identified as the potential high risk group from exposure to microbial pollution of recreational marine waters (Wade 2008; EPA 2007). In

addition to the Pilot Epidemiologic Study , the investigators have performed some pil ot studies of bather shedding in toddlers wearing diapers and plan to pursue additional studies in children in the future . By using only residents, not tourists, and in particular residents that were willing to go bathing at the Study Site, there

was some indication from the Hawaii studies (Fujioka 1994) that we may have selected for persons less susceptible to illnesses caused by exposure to microbial pollution in recreational marine waters due to self selection and the exclusion of persons with obvious im munologic susceptibility

Nevertheless we believe that our study had a number of important strengths ( Fleisher 1991, Fleisher 1993,
Fleisher 1996, Fleisher 1998, Pruss 1998, Wade 2003, Fleis her 2006, Craun 2006, EPA 2007, Boehm in . Nevertheless, as Fleisher et al found increased report of illness among their resident participants in the United Kingdom, we also found an increased risk of reported illness even among our potentially less susceptible resident b ather participants in the exposed bather group for report of gastrointestinal symptoms

.

As noted above, individual monitoring was only able to evaluate the indicator organism (enterococcus) and the pathogen, S. aureus ; due to expense and logistics, addit ional individual pathogen monitoring was not possible, only daily for the 15 study days.

### 24 press) . To our knowledge, this was the first study performed in the US as a randomized design, and the first study in the world as a randomized design in non point source sub/tropical recreational marine waters. As noted above, we believe that the randomized design is very important since it minimizes the biases contained in previous non

- randomized studies as explained above . We were also able to perform extensive individual environmental sampling in a Study Site th at had been extremely well characterized as a non point source. Finally, we were able to capture a wide variability in the indicator organism measurements as well as perform the study with a large sample size over a 7 month period with variable environment al conditions.

C ONCLUSIONS & R ECOMMENDATIONS

The results of this study demonstrate d that even non point source recreational marine waters, at least in sub/tropical areas, should be evaluated for possible exposures and health risks to human bathers.

Furt hermore, although our results indicate d some association between the currently used indicator organism, enterococcus, and reported heal th risks in human bathers, the apparent lack of direct association between the

enterococcus association and measurement o f pathogens needs to be evaluated further. Future studies should also focus on developing fate and tra nsport models, quantitative microbial risk assessments

, and epidemiologic studies which incorporate human, microbiological and oceanographic data

.

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### 29 A PPENDICES

Human Subjects ( IRB ) Approval

**•** UM

**•** FL DOH

Study Consent Form (English & Spanish)

Study Questionnaires (English & Spanish)

- Of note, in this study, we combined the 2 follow up questionnaires due to limited resources: we used the first page of the 7 day follow up and then the entire Postal Questionn aire; both questionnaires were administered over the phone in English and Spanish at day 7 after the Study

exposure.

- T he original Questionnaires were provided by Dr David Kay ( dave@crehkay.demon.co.uk ) and

ha ve been used in similarly designed studies in Europe