Exposure to Human-Associated Chemical Markers of Fecal Contamination and Self-Reported Illness among Swimmers at Recreational Beaches.

Anthropogenic chemicals have been proposed as potential markers of human fecal contamination in recreational water. However, to date, there are no published studies describing their relationships with illness risks. Using a cohort of swimmers at seven U.S. beaches, we examined potential associations between the presence of chemical markers of human fecal pollution and self-reported gastrointestinal (GI) illness, diarrhea, and respiratory illness. Swimmers were surveyed about their beach activities, water exposure, and baseline symptoms on the day of their beach visit, and about any illness experienced 10-12 days later. Risk differences were estimated using model-based standardization and adjusted for the swimmer's age, beach site, sand contact, rainfall, and water temperature. Sixty-two chemical markers were analyzed from daily water samples at freshwater and marine beaches. Of those, 20 were found consistently. With the possible exception of bisphenol A and cholesterol, no chemicals were consistently associated with increased risks of illness. These two chemicals were suggestively associated with 2% and 1% increased risks of GI illness and diarrhea in both freshwater and marine beaches. Additional research using the more sensitive analytic methods currently available for a wider suite of analytes is needed to support the use of chemical biomarkers to quantify illness risk and identify fecal pollution sources.

Coliphages and Gastrointestinal Illness in Recreational Waters: Pooled Analysis of Six Coastal Beach Cohorts.

BACKGROUND: Coliphages have been proposed as indicators of fecal contamination in recreational waters because they better mimic the persistence of pathogenic viruses in the environment and wastewater treatment than fecal indicator bacteria. We estimated the association between coliphages and gastrointestinal illness and compared it with the association with culturable enterococci. METHODS: We pooled data from six prospective cohort studies that enrolled coastal beachgoers in California, Alabama, and Rhode Island. Water samples were collected and gastrointestinal illness within 10 days of the beach visit was recorded. Samples were tested for enterococci and male-specific and somatic coliphages. We estimated cumulative incidence ratios (CIR) for the association between swimming in water with detectable coliphage and gastrointestinal illness when human fecal pollution was likely present, not likely present, and under all conditions combined. The reference group was unexposed swimmers. We defined continuous and threshold-based exposures (coliphage present/absent, enterococci >35 vs. ≤35 CFU/100 ml). RESULTS: Under all conditions combined, there was no association between gastrointestinal illness and swimming in water with detectable coliphage or enterococci. When human fecal pollution was likely present, coliphage and enterococci were associated with increased gastrointestinal illness, and there was an association between male-specific coliphage level and illness that was somewhat stronger than the association between enterococci and illness. There were no substantial differences between male-specific and somatic coliphage. CONCLUSIONS: Somatic coliphage and enterococci had similar associations with gastrointestinal illness; there was some evidence that male-specific coliphage had a stronger association with illness than enterococci in marine waters with human fecal contamination.

Exposure to human-associated fecal indicators and self-reported illness among swimmers at recreational beaches: a cohort study.

BACKGROUND: Fecal indicator bacteria used to assess illness risks in recreational waters (e.g., Escherichia coli, Enterococci) cannot discriminate among pollution sources. To address this limitation, human-associated Bacteroides markers have been proposed, but the risk of illness associated with the presence of these markers in recreational waters is unclear. Our objective was to estimate associations between human-associated Bacteroides markers in water and self-reported illness among swimmers at 6 U.S. beaches spanning 2003-2007. METHODS: We used data from a prospectively-enrolled cohort of 12,060 swimmers surveyed about beach activities and water exposure on the day of their beach visit. Ten to twelve days later, participants reported gastroinestinal, diarrheal, and respiratory illnesses experienced since the visit. Daily water samples were analyzed for the presence of human-associated Bacteroides genetic markers: HF183, BsteriF1, BuniF2, HumM2. We used model-based standardization to estimate risk differences (RD) and 95% confidence intervals (CI). We assessed whether the presence of Bacteroides markers were modifiers of the association between general Enterococcus and illness among swimmers using interaction contrast. RESULTS: Overall we observed inconsistent associations between the presence of Bacteroides markers and illness. There was a pattern of increased risks of gastrointestinal (RD = 1.9%; 95% CI: 0.1%, 3.7%), diarrheal (RD = 1.3%; 95% CI: -0.2%, 2.7%), and respiratory illnesses (RD = 1.1%; 95% CI: -0.2%, 2.5%) associated with BsteriF1. There was no evidence that Bacteroides markers acted as modifiers of Enterococcus and illness. Patterns were similar when stratified by water matrix. CONCLUSIONS: Quantitative measures of fecal pollution using Bacteroides, rather than presence-absence indicators, may be necessary to accurately assess human risk specific to the presence of human fecal pollution.

Acute Gastroenteritis and Recreational Water: Highest Burden Among Young US Children.

OBJECTIVES: To provide summary estimates of gastroenteritis risks and illness burden associated with recreational water exposure and determine whether children have higher risks and burden. METHODS: We combined individual participant data from 13 prospective cohorts at marine and freshwater beaches throughout the United States (n = 84 411). We measured incident outcomes within 10 days of exposure: diarrhea, gastrointestinal illness, missed daily activity (work, school, vacation), and medical visits. We estimated the relationship between outcomes and 2 exposures: body immersion swimming and Enterococcus spp. fecal indicator bacteria levels in the water. We also estimated the population-attributable risk associated with these exposures. RESULTS: Water exposure accounted for 21% of diarrhea episodes and 9% of missed daily activities but was unassociated with gastroenteritis leading to medical consultation. Children aged 0 to 4 and 5 to 10 years had the most water exposure, exhibited stronger associations between levels of water quality and illness, and accounted for the largest attributable illness burden. CONCLUSIONS: The higher gastroenteritis risk and associated burden in young children presents important new information to inform future recreational water quality guidelines designed to protect public health.

Swimming in the USA: beachgoer characteristics and health outcomes at US marine and freshwater beaches.

Swimming in lakes and oceans is popular, but little is known about the demographic characteristics, behaviors, and health risks of beachgoers on a national level. Data from a prospective cohort study of beachgoers at multiple marine and freshwater beaches in the USA were used to describe beachgoer characteristics and health outcomes for swimmers and non-swimmers. This analysis included 54,250 participants. Most (73.2%) entered the water; of those, 65.1% put their head under water, 41.3% got water in their mouth and 18.5% swallowed water. Overall, 16.3% of beachgoers reported any new health problem. Among swimmers, 6.6% reported gastrointestinal (GI) illness compared with 5.5% of non-swimmers (unadjusted χ² p < 0.001); 6.0% of swimmers and 4.9% of non-swimmers reported respiratory illness (p < 0.001); 1.8% of swimmers and 1.0% of non-swimmers reported ear problems (p < 0.001); and 3.9% of swimmers and 2.4% of non-swimmers experienced a rash (p < 0.001). Overall, swimmers reported a higher unadjusted incidence of GI illness and earaches than non-swimmers. Current surveillance systems might not detect individual cases and outbreaks of illness associated with swimming in natural water. Better knowledge of beachgoer characteristics, activities, and health risks associated with swimming in natural water can improve disease surveillance and prioritize limited resources.

Assessment of swimmer behaviors on pool water ingestion.

Enteric pathogens in pool water can be unintentionally ingested during swimming, increasing the likelihood of acute gastrointestinal illness (AGI). AGI cases in outbreaks are more likely to submerge heads than non-cases, but an association is unknown since outbreak data are self-reported and prone to bias. In the present study, head submersion frequency and duration were observed and analyzed for associations with pool water ingestion measured using ultra high pressure liquid chromatography - tandem mass spectrometry. Frequency of splashes to the face was also quantified. Reliable tools that assess activities associated with pool water ingestion are needed to identify ingestion risk factors and at-risk populations. Objectives were to determine if the observed activities were associated with ingestion, and to test environmental sensor and videography assessment tools. Greater frequency and duration of head submersion were not associated with ingestion, but frequency of splashes to the face, leisurely swimming, and being ≤18 were. Videography was validated for assessing swimmer head submersion frequency. Results demonstrate ingestion risk factors can be identified using videography and urine analysis techniques. Expanding surveys to include questions on leisure swimming participation and frequency of splashes to the face is recommended to improve exposure assessment during outbreak investigations.

The incidence and health burden of earaches attributable to recreational swimming in natural waters: a prospective cohort study.

BACKGROUND: Earaches and outer ear infections are commonly associated with swimming. In this study, we estimated the excess risk and health burden of earaches due to swimming in natural fresh and marine waters using results from a survey of over 50,000 beachgoers at nine beaches across the United States. METHODS: Prospective cohort studies were conducted at four freshwater and five marine sites in the United States and Puerto Rico. Beach visitors were enrolled on summer weekends and holidays. Ten to twelve days after the beach visit, respondents answered questions about health symptoms, including earaches or ear infections experienced since the beach visit. Economic and physical burdens were also obtained. Fixed slope, random intercept (beach site) multivariate logistic regression models were used to estimate the relationship between head immersion swimming exposure and earaches. Model results were used to calculate excess risk for earaches attributable to swimming. RESULTS: The overall incidence of self-reported earache was 1.6% in the 10-12 days after the beach visit. Earaches were more frequent in head immersion swimmers compared to non-swimmers for all beach sites and age groups. Earaches were unassociated with water sample measures of fecal contamination and turbidity. After adjustment for covariates, we calculated 7.12 excess earaches among head immersion swimmers per 1,000 swimming events. Twenty-four percent of those with earache reported missing their regular activities; 28% visited a doctor; 4% visited the emergency room; and 31% and 40% used prescription and non-prescription medications, respectively. CONCLUSIONS: There are at least 128 million swimming events in natural waters annually. Such frequent exposures could result in 900,000 excess earaches, 260,000 visits to the doctor, 39,900 visits to the emergency room, nearly $4 million dollars in out-of-pocket expenditures on prescription and over-the-counter medications, and close to 75,000 hours of clinician time. More accurate estimates of swimming exposure are needed to improve population burden and associated cost estimates.

Equivalency of risk for a modified health endpoint: a case from recreational water epidemiology studies.

BACKGROUND: The United States Environmental Protection Agency (USEPA) and its predecessors have conducted three distinct series of epidemiological studies beginning in 1948 on the relationship between bathing water quality and swimmers' illnesses. Keeping pace with advances in microbial technologies, these studies differed in their respective microbial indicators of water quality. Another difference, however, has been their specific health endpoints. The latest round of studies, the National Epidemiological Assessment of Recreational (NEEAR) Water studies initiated in 2002, used a case definition, termed "NEEAR GI illness" (NGI), for gastrointestinal illness corresponding closely to classifications employed by contemporary researchers, and to that proposed by the World Health Organization. NGI differed from the previous definition of "highly credible gastrointestinal illness" (HCGI) upon which the USEPA's 1986 bathing water criteria had been based, primarily by excluding fever as a prerequisite. METHODS: Incidence of NGI from the NEEAR studies was compared to that of HCGI from earlier studies. Markov chain Monte Carlo method was used to estimate the respective beta binomial probability densities for NGI and HCGI establish credible intervals for the risk ratio of NGI to HCGI. RESULTS: The ratio of NGI risk to that of HCGI is estimated to be 4.5 with a credible interval 3.2 to 7.7. CONCLUSIONS: A risk level of 8 HCGI illnesses per 1000 swimmers, as in the 1986 freshwater criteria, would correspond to 36 NGI illnesses per 1000 swimmers. Given a microbial DNA-based (qPCR) water quality vs. risk relationship developed from the NEEAR studies, 36 NGI per 1000 corresponds to a geometric mean of 475 qPCR cell-equivalents per 100 ml.

Comparison of the multiple-sample means with composite sample results for fecal indicator bacteria by quantitative PCR and culture.

Few studies have addressed the efficacy of composite sampling for measuring indicator bacteria by quantitative PCR (qPCR). We compared results from composited samples with multiple-sample means for culture- and qPCR-based water quality monitoring. Results from composited samples for both methods were similarly correlated to multiple-sample means and predicted criteria exceedances equally.

Fecal indicators in sand, sand contact, and risk of enteric illness among beachgoers.

BACKGROUND: Beach sand can harbor fecal indicator organisms and pathogens, but enteric illness risk associated with sand contact remains unclear. METHODS: In 2007, visitors at 2 recreational marine beaches were asked on the day of their visit about sand contact. Ten to 12 days later, participants answered questions about health symptoms since the visit. F+ coliphage, Enterococcus, Bacteroidales, fecal Bacteroides, and Clostridium spp. in wet sand were measured using culture and molecular methods. RESULTS: We analyzed 144 wet sand samples and completed 4999 interviews. Adjusted odds ratios (aORs) were computed, comparing those in the highest tertile of fecal indicator exposure with those who reported no sand contact. Among those digging in sand compared with those not digging in sand, a molecular measure of Enterococcus spp. (calibrator cell equivalents/g) in sand was positively associated with gastrointestinal (GI) illness (aOR = 2.0 [95% confidence interval (CI) = 1.2-3.2]) and diarrhea (2.4 [1.4-4.2]). Among those buried in sand, point estimates were greater for GI illness (3.3 [1.3-7.9]) and diarrhea (4.9 [1.8-13]). Positive associations were also observed for culture-based Enterococcus (colony-forming units/g) with GI illness (aOR digging = 1.7 [1.1-2.7]) and diarrhea (2.1 [1.3-3.4]). Associations were not found among nonswimmers with sand exposure. CONCLUSIONS: We observed a positive relationship between sand-contact activities and enteric illness as a function of concentrations of fecal microbial pollution in beach sand.

Dramatic improvements in beach water quality following gull removal.

Gulls are often cited as important contributors of fecal contamination to surface waters, and some recreational beaches have used gull control measures to improve microbial water quality. In this study, gulls were chased from a Lake Michigan beach using specially trained dogs, and water quality improvements were quantified. Fecal indicator bacteria and potentially pathogenic bacteria were measured before and during gull control using culture methods and quantitative polymerase chain reaction (qPCR). Harassment by dogs was an effective method of gull control: average daily gull populations fell from 665 before to 17 during intervention; and a significant reduction in the density of a gull-associated marker was observed (p < 0.001). Enterococcus spp. and Escherichia coli densities were also significantly reduced during gull control (p < 0.001 and p = 0.012, respectively for culture methods; p = 0.012 and p = 0.034, respectively for qPCR). Linear regression results indicate that a 50% reduction in gulls was associated with a 38% and 29% decrease in Enterococcus spp. and E. coli densities, respectively. Potentially human pathogenic bacteria were detected on 64% of days prior to gull control and absent during gull intervention, a significant reduction (p = 0.005). This study demonstrates that gull removal can be a highly successful beach remedial action to improve microbial water quality.

Health risks to children from exposure to fecally-contaminated recreational water.

BACKGROUND: Children may be at higher risk for swimming-associated illness following exposure to fecally-contaminated recreational waters. We analyzed a pooled data set of over 80,000 beachgoers from 13 beach sites across the United States to compare risks associated with the fecal indicator bacteria Enterococcus spp. (measured by colony forming units, CFU and quantitative polymerase chain reaction cell equivalents, qPCR CE) for different age groups across different exposures, sites and health endpoints. METHODS: Sites were categorized according to the predominant type of fecal contamination (human or non-human). Swimming exposures of varying intensity were considered according to degree of contact and time spent in the water. Health endpoints included gastrointestinal and respiratory symptoms and skin rashes. Logistic regression models were used to analyze the risk of illness as a function of fecal contamination in water as measured by Enterococcus spp. among the exposed groups. Non-swimmers (those who did not enter the water) were excluded from the models to reduce bias and facilitate comparison across groups. RESULTS: Gastrointestinal symptoms were the most sensitive health endpoint and strongest associations were observed with Enterococcus qPCR CE at sites impacted by human fecal contamination. Under several exposure scenarios, associations between illness and Enterococcus spp. levels were significantly higher among children compared to adolescents and adults. Respiratory symptoms were also associated with Enterococcus spp. exposures among young children at sites affected by human fecal sources, although small sample sizes resulted in imprecise estimates for these associations. CONCLUSION: Under many exposure scenarios, children were at higher risk of illness associated with exposure to fecal contamination as measured by the indicator bacteria Enterococcus spp. The source of fecal contamination and the intensity of swimming exposure were also important factors affecting the association between Enterococcus spp. and swimming-associated illness.

Comparative stability of assay results of enterococci measured by culture and qPCR over time in bathing beach waters.

The diurnal presence of the culturable bacterial indicators of fecal contamination in the water environment has been shown to be highly variable over time due to natural die-off and injury from effects of sunlight and other environmental stressors. Molecular analytes of a quantitative polymerase chain reaction (qPCR) method for measuring fecal contamination degrade considerably slower than the alternative of culturable fecal indicator bacteria. The rapid qPCR method holds the promise of more timely notification decisions with respect to postings or closure being made on the basis of microbial water quality samples collected earlier on the same day. In the case of culture-based methods requiring a 24 h or longer incubation period, decisions must be based on samples collected no sooner than the previous day. To examine the effect of this lag in assay results, temporal stability of a molecular Enterococci target analyte with that of traditional culture-based cells is compared using data from USEPA studies conducted between 2003 and 2007 on seven freshwater and marine beaches that were impacted by publicly-owned treatment works. Generally, levels of the molecular indicator were more consistent throughout the day between 8:00 am and 3:00 pm. The difference in temporal consistency is even more pronounced when the 24-h lag in culture-based results is taken into account.

Rapidly measured indicators of recreational water quality and swimming-associated illness at marine beaches: a prospective cohort study.

INTRODUCTION: In the United States and elsewhere, recreational water quality is monitored for fecal indicator bacteria to help prevent swimming-associated illnesses. Standard methods to measure these bacteria take at least 24 hours to obtain results. Molecular approaches such as quantitative polymerase chain reaction (qPCR) can estimate these bacteria faster, in under 3 hours. Previously, we demonstrated that measurements of the fecal indicator bacteria Enterococcus using qPCR were associated with gastrointestinal (GI) illness among swimmers at freshwater beaches. In this paper, we report on results from three marine beach sites. METHODS: We interviewed beach-goers and collected water samples at marine beaches affected by treated sewage discharges in Mississippi in 2005, and Rhode Island and Alabama in 2007. Ten to twelve days later, we obtained information about gastrointestinal, respiratory, eye, ear and skin symptoms by telephone. We tested water samples for fecal indicator organisms using qPCR and other methods. RESULTS: We enrolled 6,350 beach-goers. The occurrence of GI illness among swimmers was associated with a log10-increase in exposure to qPCR-determined estimates of fecal indicator organisms in the genus Enterococcus (AOR = 2.6, 95% CI 1.3-5.1) and order Bacteroidales (AOR = 1.9, 95% CI 1.3-2.9). Estimates of organisms related to Clostridium perfringens and a subgroup of organisms in the genus Bacteroides were also determined by qPCR in 2007, as was F+ coliphage, but relationships between these indicators and illness were not statistically significant. CONCLUSIONS: This study provides the first evidence of a relationship between gastrointestinal illness and estimates of fecal indicator organisms determined by qPCR at marine beaches.

Evaluating health risks associated with exposure to ambient surface waters during recreational activities: A systematic review and meta-analysis.

Recreational water quality guidelines protect the public from health risks associated with water recreation by helping to prevent unacceptable concentrations of pathogenic organisms in ambient water. However, illness risk is associated with both the concentration of pathogens in the water and the degree of contact with those pathogens. Different recreational activities can result in different levels of contact with ambient water containing water-borne pathogens. We conducted a systematic literature review and meta-analysis to evaluate risks of illness associated with different recreational activities and different levels of contact to ambient surface waters. We screened 8,618 potentially relevant studies for quantitative measures of risk using inclusion/exclusion criteria established in advance. We categorized recreational activities as swimming, sports-related contact, minimal contact, and sand contact. We combined relative risks using a random effects meta-analysis for adverse health outcome categories representing gastrointestinal illness, respiratory illness, skin, eye, ear, nose, throat, and cold/flu illness. We identified 92 studies meeting our inclusion criteria. Pooled risk estimates indicate significant elevation of gastrointestinal illness with the recreational activity categories swimming (2.19, 95% CI: 1.82, 2.63) and sports-related contact (2.69, 95% CI: 1.04, 6.92), and nonsignificant elevation of gastrointestinal illness with minimal contact (1.27, 95% CI: 0.74, 2.16). We also found a significant elevation of respiratory illness with swimming (1.78, 95% CI: 1.38, 2.29) and sports-related contact (1.49, 95% CI: 1.00, 2.24), and no elevation of respiratory illness with minimal contact (0.90, 95% CI: 0.71, 1.14). This study suggests that exposures associated with different types of recreational activities are important characteristics of the exposure pathway when assessing illness risk associated with recreation in ambient surface waters.

Contact with beach sand among beachgoers and risk of illness.

Recent studies of beach sand fecal contamination have triggered interest among scientists and in the media. Although evidence shows that beach sand can harbor high concentrations of fecal indicator organisms, as well as fecal pathogens, illness risk associated with beach sand contact is not well understood. Beach visitors at 7 US beaches were enrolled in the National Epidemiological and Environmental Assessment of Recreational Water (NEEAR) Study during 2003-2005 and 2007 and asked about sand contact on the day of their visit to the beach (digging in the sand, body buried in the sand). Then, 10-12 days after their visit, participants were telephoned to answer questions about any health symptoms experienced since the visit. The authors completed 27,365 interviews. Digging in the sand was positively associated with gastrointestinal illness (adjusted incidence proportion ratio (aIPR) = 1.13, 95% confidence interval (CI): 1.02, 1.25) and diarrhea (aIPR = 1.20, 95% CI: 1.05, 1.36). The association was stronger between those buried in the sand and gastrointestinal illness (aIPR = 1.23, 95% CI: 1.05, 1.43) and diarrhea (aIPR = 1.24, 95% CI: 1.01, 1.52). Nonenteric illnesses did not show a consistent association with sand contact activities. Sand contact activities were associated with enteric illness at beach sites. Variation in beach-specific results suggests that site-specific factors may be important in the risk of illness following sand exposure.

Application of a multiplex salivary immunoassay to detect sporadic incident norovirus infections.

Norovirus is one of the most common causes of gastroenteritis. Following infection, anti-norovirus salivary immunoglobulin G (IgG) rises steeply within 2 weeks and remains elevated for several months; this immunoconversion can serve as an indicator of infection. We used a multiplex salivary immunoassay to study norovirus infections among 483 visitors to a Lake Michigan beach in 2015. Saliva was collected on the day of the beach visit (S1); after 10-14 days (S2); and after 30-40 days (S3). Luminex microspheres were coupled to recombinant antigens of genogroup I (GI) and II (GII) noroviruses and incubated with saliva. Immunoconversion was defined as at least 4-fold increase in anti-norovirus IgG antibody response from S1 to S2 and a 3-fold increase from S1 to S3. Ten (2.1%) immunoconverted to either GI (2) or GII (8) norovirus. Among those who immunoconverted, 40% reported at least one gastrointestinal symptom and 33% reported diarrhea, compared to 15% (p = 0.06) and 8% (p = 0.04) among those who did not immunoconvert, respectively. The two participants who immunoconverted to GI norovirus both swallowed water during swimming (p = 0.08). This study demonstrated the utility of a non-invasive salivary immunoassay to detect norovirus infections and an efficient approach to study infectious agents in large cohorts.

Child environmental exposures to water and sand at the beach: Findings from studies of over 68,000 subjects at 12 beaches.

Swimming and recreating in lakes, oceans, and rivers is common, yet the literature suggests children may be at greater risk of illness following such exposures. These effects might be due to differences in immunity or differing behavioral factors such as poorer hygiene, longer exposures to, and greater ingestion of potentially contaminated water and sand. We pooled data from 12 prospective cohorts (n=68,685) to examine exposures to potentially contaminated media such as beach water and sand among children compared with adults, and conducted a simulation using self-reported time spent in the water and volume of water swallowed per minute by age to estimate the total volume of water swallowed per swimming event by age category. Children aged 4-7 and 8-12 years had the highest exposures to water, sand, and algae compared with other age groups. Based on our simulation, we found that children (6-12 years) swallow a median of 36 ml (90th percentile=150 ml), whereas adults aged ≥35 years swallow 9 ml (90th percentile=64 ml) per swimming event, with male children swallowing a greater amount of water compared with females. These estimates may help to reduce uncertainty surrounding routes and durations of recreational exposures and can support the development of chemical and microbial risk assessments.

Asymptomatic norovirus infection associated with swimming at a tropical beach: A prospective cohort study.

BACKGROUND: Swimming in fecally-contaminated waterbodies can result in gastrointestinal infections. However, the pathogenic microorganisms responsible are not well understood because sporadic cases of illness are not reported completely, exposure information is often not collected, and epidemiology studies rely on self-reported symptoms. Noroviruses are considered a likely cause because they are found in high densities in sewage, resistant to wastewater treatment and survive in the environment. In this study, saliva samples were collected from subjects at a beach in Puerto Rico and tested for evidence of norovirus-specific IgG responses as an indicator of incident norovirus infection. METHODS: Saliva samples were collected from 1298 participants using an oral swab. Samples were collected on the day of the beach visit (S1); after 10-12 days (S2); and after three weeks (S3). Saliva was tested for IgG responses to GI.1 and GII.4 noroviruses using a microsphere based multiplex salivary immunoassay. Immunoconversion was defined as a four-fold increase in median fluorescence intensity (MFI) from S1 to S2 with the S3 sample at least three times above the S1 MFI. RESULTS: Thirty-four subjects (2.6%) immunoconverted to GI.1 or GII.4 norovirus. Swimmers who immersed their head in water had a higher rate of immunoconversion (3.4%), compared to either non-swimmers (0.0%, p = 0.003) or waders and non-swimmers combined (0.4%, Odds Ratio: 5.07, 95% Confidence Interval:1.48-17.00). Immunoconversion was not associated with gastrointestinal symptoms. CONCLUSIONS: This is the first study to demonstrate an association between swimming at a beach impacted by fecal contamination and asymptomatic norovirus infection. The findings implicate recreational water as potentially important transmission pathway for norovirus infection.

Integrated preservation and sample clean up procedures for studying water ingestion by recreational swimmers via urinary biomarker determination.

The use of cyanuric acid as a biomarker for ingestion of swimming pool water may lead to quantitative knowledge of the volume of water ingested during swimming, contributing to a better understanding of disease resulting from ingestion of environmental contaminants. When swimming pool water containing chlorinated cyanurates is inadvertently ingested, cyanuric acid is excreted quantitatively within 24 h as a urinary biomarker of ingestion. Because the volume of water ingested can be quantitatively estimated by calculation from the concentration of cyanuric acid in 24 h urine samples, a procedure for preservation, cleanup, and analysis of cyanuric acid was developed to meet the logistical demands of large scale studies. From a practical stand point, urine collected from swimmers cannot be analyzed immediately, given requirements of sample collection, shipping, handling, etc. Thus, to maintain quality control to allow confidence in the results, it is necessary to preserve the samples in a manner that ensures as quantitative analysis as possible. The preservation and clean-up of cyanuric acid in urine is complicated because typical approaches often are incompatible with the keto-enol tautomerization of cyanuric acid, interfering with cyanuric acid sample preparation, chromatography, and detection. Therefore, this paper presents a novel integration of sample preservation, clean-up, chromatography, and detection to determine cyanuric acid in 24 h urine samples. Fortification of urine with cyanuric acid (0.3-3.0 mg/L) demonstrated accuracy (86-93% recovery) and high reproducibility (RSD < 7%). Holding time studies in unpreserved urine suggested sufficient cyanuric acid stability for sample collection procedures, while longer holding times suggested instability of the unpreserved urine. Preserved urine exhibited a loss of around 0.5% after 22 days at refrigerated storage conditions of 4 °C.