Detection of enteric viruses and SARS-CoV-2 in beach sand.

Beach sand harbors a diverse group of microbial organisms that may be of public health concern. Nonetheless, little is known about the presence and distribution of viruses in beach sand. In this study, the first objective was to evaluate the presence of seven viruses (Aichi virus, enterovirus, hepatitis A virus, human adenovirus, norovirus, rotavirus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)) in sands collected at public beaches. The second objective was to assess the spatial distribution of enteric viruses in beach sand. To that end, 27 beach sand samples from different beaches in Portugal were collected between November 2018 and August 2020 and analyzed for the presence of viruses. At seven beaches, samples were collected in the supratidal and intertidal zones. Results show that viruses were detected in 89 % (24/27) of the sand samples. Aichi virus was the most prevalent (74 %). Noroviruses were present in 19 % of the samples (norovirus GI - 15 %, norovirus GII - 4 %). Human adenovirus and enterovirus were detected in 48 % and 22 % of the samples, respectively. Hepatitis A virus and rotavirus were not detected. Similarly, SARS-CoV-2 in beach sand collected during the initial stages of the pandemic was also not detected. The detection of three or more viruses occurred in 15 % of the samples. Concentrations of viruses were as high as 7.2 log copies (cp)/g of sand. Enteric viruses were found in higher prevalence in sand collected from the supratidal zone compared to the intertidal zone. Human adenovirus was detected in 43 % of the supratidal and 14 % in the intertidal samples and Aichi virus in 57 % and 86 % of the intertidal and supratidal areas, respectively. Our findings suggest that beach sand can be a reservoir of enteric viruses, suggesting that it might be a vehicle for disease transmission, particularly for children, the elderly, and immunocompromised users.

Impact of wastewater infrastructure improvements on beach water fecal indicator bacteria levels in Monroe County, Florida.

The effects of wastewater infrastructure construction on regional and local environments is unknown. This project evaluated the effects of such projects in Monroe County, Florida, an area that had undergone regional wastewater infrastructure improvements. We used fecal indicator bacteria (FIB) (fecal coliform and enterococci), as a proxy indicator of beach water quality for an 18-year period of record. At the highest level of aggregation, FIBs for all 17 beaches within the county were combined to evaluate trends on a yearly basis. At the lower level, yearly FIB trends were evaluated for each beach separately. FIB data on infrastructure project period (categorical variables: before, during, and after construction), and the influences of environmental conditions (quantitative variables of rainfall and temperature) were also evaluated. In the multiple regression models, enterococci and fecal coliform were significantly associated with rainfall (24 h, p < 0.0001) and water temperature (p < 0.0001) when only the quantitative variables were considered. When both categorical and quantitative variables were considered, project period was significant for enterococci (p < 0.0001) and fecal coliform (p < 0.0001), as was 24 h lagged rainfall. Overall, the most significant factors for both fecal coliform and enterococci were rainfall and project period. Considering all beaches, infrastructure projects seem to have the collective desired effects in the years following construction, as there were decreased FIBs measured at beach sites. Only through the aggregation of all projects and measurements at all beach sites could the decreases in FIB levels be observed. Local analysis is needed to explain anomalies from these general trends for specific beaches. This understanding of FIBs, their responses to environmental and project factors, and the need for aggregated and local site analysis can provide guidance to managers at other locations with similar issues of failing wastewater infrastructure and frequent FIB exceedances.

Untreated sewage contamination of beach sand from a leaking underground sewage system.

Thirty people (mostly children) experienced an episode of skin rash days after a sand sifting beach operation at Porto Pim Beach in Faial, Azores during June 2019. An environmental and epidemiologic investigation was conducted to identify the cause of the outbreak of skin rash. The epidemiologic investigation found that some of the patients experiencing symptoms had never entered the beach water. During the pollution period and throughout the epidemiologic investigation, faecal indicator bacteria levels (94 CFU/100 ml for intestinal enterococci and 61 CFU/100 ml for Escherichia coli) in water remained under the limits used for the ninety-five percentile calculation of an Excellent coastal and transitional bathing water defined in the Portuguese Legislation (100 CFU/100 ml for intestinal enterococci and 250 CFU/100 ml for Escherichia coli). Thus sand contact was considered as a likely primary exposure route. Sand microbiological analysis for faecal indicator organisms and electron microscopy strongly suggested faecal contamination. Chemical analysis of the sand also revealed a concomitant substance compatible with sodium-hypochlorite as analysed using gas chromatography and subsequently confirmed by free chlorine analysis. Inspection of the toilet facilities and sewage disposal system revealed a leaking sewage distribution box. Collectively, results suggest that the cause of the outbreak was the leaking underground sewage distribution box that serviced the beach toilet facilities (40 m from beach), where sodium-hypochlorite was used for cleaning and disinfection. This sewage then contaminated the surficial sands to which beach goers were exposed. Chlorine being an irritant substance, was believed to have been the cause of the symptoms given the sudden presentation and dissipation of skin rashes. No gastro-intestinal illness was reported during this episode and during the following 30 days. Like water, beach sand should also be monitored for safety, especially for areas serviced by aged infrastructure.

Proliferation of microalgae and enterococci in the Lake Okeechobee, St. Lucie, and Loxahatchee watersheds.

This study is an analysis of relationships between microalgae (measured as chlorophyll a) and the fecal indicator bacteria enterococci. Microalgae blooms and enterococci exceedances have been occurring in Florida's recreational waterways for years. More recently, this has become a management concern as microalgae blooms have been attributed to potentially toxic cyanobacteria, and enterococci exceedances link to human infection/illness. Since both the microalgal blooms and bacterial exceedances occur in regions that receive managed freshwater releases from Lake Okeechobee, we hypothesized that both the blooms and exceedances are related to excess nutrients from the lake. Two experimental sites, on Lake Okeechobee and the St. Lucie River (downstream of the lake), plus a control site on the Loxahatchee River (which does not receive lake flow) were evaluated. The hypothesis was evaluated through three study components: 1) analysis of available long-term data from local environmental databases, 2) a year-long monthly sampling and analysis of chlorophyll a, enterococci, nutrients, and physical-chemical data, and 3) microcosm experiments with altered water/sediment conditions. Results support the hypothesis that excess nutrients play a role in both chlorophyll a and enterococci levels. For the St. Lucie River, analyses indicate that chlorophyll a correlated significantly with total Kjeldahl nitrogen (TKN) (R2 = 0.30, p = 0.008) and the strongest model for enterococci included nitrate-nitrite, TKN, total phosphorus, orthophosphorus, and turbidity in our long-term analysis (n = 39, R2 = 0.83, p ≤ 0.001). The microcosm results indicated that chlorophyll a and enterococci only persisted for 36 h in water from all sources, and that sediments from Lake Okeechobee may have allowed for sustained levels of chlorophyll a and enterococci levels. Overall similarities were observed in chlorophyll a and enterococci relationships with nutrient concentrations regardless of a Lake Okeechobee connection, as underscored by a study of flow out of the lake and downstream areas. This suggests that both nutrient-rich lake water and untreated surface water runoff contribute to microalgae blooms and enterococci exceedances in southeast Florida.

Routine screening of harmful microorganisms in beach sands: implications to public health.

Beaches worldwide provide recreational opportunities to hundreds of millions of people and serve as important components of coastal economies. Beach water is often monitored for microbiological quality to detect the presence of indicators of human sewage contamination so as to prevent public health outbreaks associated with water contact. However, growing evidence suggests that beach sand can harbor microbes harmful to human health, often in concentrations greater than the beach water. Currently, there are no standards for monitoring, sampling, analyzing, or managing beach sand quality. In addition to indicator microbes, growing evidence has identified pathogenic bacteria, viruses, and fungi in a variety of beach sands worldwide. The public health threat associated with these populations through direct and indirect contact is unknown because so little research has been conducted relating to health outcomes associated with sand quality. In this manuscript, we present the consensus findings of a workshop of experts convened in Lisbon, Portugal to discuss the current state of knowledge on beach sand microbiological quality and to develop suggestions for standardizing the evaluation of sand at coastal beaches. The expert group at the "Microareias 2012" workshop recommends that 1) beach sand should be screened for a variety of pathogens harmful to human health, and sand monitoring should then be initiated alongside regular water monitoring; 2) sampling and analysis protocols should be standardized to allow proper comparisons among beach locations; and 3) further studies are needed to estimate human health risk with exposure to contaminated beach sand. Much of the manuscript is focused on research specific to Portugal, but similar results have been found elsewhere, and the findings have worldwide implications.

Indicator microbes correlate with pathogenic bacteria, yeasts and helminthes in sand at a subtropical recreational beach site.

AIMS: Research into the relationship between pathogens, faecal indicator microbes and environmental factors in beach sand has been limited, yet vital to the understanding of the microbial relationship between sand and the water column and to the improvement of criteria for better human health protection at beaches. The objectives of this study were to evaluate the presence and distribution of pathogens in various zones of beach sand (subtidal, intertidal and supratidal) and to assess their relationship with environmental parameters and indicator microbes at a non-point source subtropical marine beach. METHODS AND RESULTS: In this exploratory study in subtropical Miami (Florida, USA), beach sand samples were collected and analysed over the course of 6 days for several pathogens, microbial source tracking markers and indicator microbes. An inverse correlation between moisture content and most indicator microbes was found. Significant associations were identified between some indicator microbes and pathogens (such as nematode larvae and yeasts in the genus Candida), which are from classes of microbes that are rarely evaluated in the context of recreational beach use. CONCLUSIONS: Results indicate that indicator microbes may predict the presence of some of the pathogens, in particular helminthes, yeasts and the bacterial pathogen Staphylococcus aureus including methicillin-resistant forms. Indicator microbes may thus be useful for monitoring beach sand and water quality at non-point source beaches. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of both indicator microbes and pathogens in beach sand provides one possible explanation for human health effects reported at non-point sources beaches.

The inter-tidal zone is the pathway of input of enterococci to a subtropical recreational marine beach.

Efforts were made to evaluate the source of enterococci levels at a recreational beach. Four monitoring efforts were implemented which included tidal studies, hourly sampling, runoff sampling, and spatially intensive sediment sampling. Spatially intensive sediment sampling indicated that enterococci concentrations consistently decreased away from the inter-tidal zone, both seaward and landward. During dry conditions, the highest concentrations in the water were observed during high tide (71±48 CFU/100 mL) and lower concentrations were observed during low tide (4±3 CFU/100 mL). Runoff was characterised by very high levels (11,700 CFU/100 mL). Results from these monitoring efforts collectively showed that the source of enterococci to the study beach is geographically located within the inter-tidal zone. Wash-in from the inter-tidal zone through tidal action and runoff plays a major role in controlling enterococci levels within the water column. Such results are significant in identifying the source and transport mechanisms of enterococci, which can subsequently be used as part of a modelling effort aimed at predicting enterococci levels at recreational beaches.

Prominent human health impacts from several marine microbes: history, ecology, and public health implications.

This paper overviews several examples of important public health impacts by marine microbes and directs readers to the extensive literature germane to these maladies. These examples include three types of dinoflagellates (Gambierdiscus spp., Karenia brevis, and Alexandrium fundyense), BMAA-producing cyanobacteria, and infectious microbes. The dinoflagellates are responsible for ciguatera fish poisoning, neurotoxic shellfish poisoning, and paralytic shellfish poisoning, respectively, that have plagued coastal populations over time. Research interest on the potential for marine cyanobacteria to contribute BMAA into human food supplies has been derived by BMAA's discovery in cycad seeds and subsequent implication as the putative cause of amyotrophic lateral sclerosis/parkinsonism dementia complex among the Chamorro people of Guam. Recent UPLC/MS analyses indicate that recent reports that BMAA is prolifically distributed among marine cyanobacteria at high concentrations may be due to analyte misidentification in the analytical protocols being applied for BMAA. Common infectious microbes (including enterovirus, norovirus, Salmonella, Campylobacter, Shigella, Staphylococcus aureus, Cryptosporidium, and Giardia) cause gastrointestinal and skin-related illness. These microbes can be introduced from external human and animal sources, or they can be indigenous to the marine environment.

Simultaneous concentration of Enterococci and coliphage from marine waters using a dual layer filtration system.

Concentrating diverse microbes in a time and cost effective manner is an essential component in water quality monitoring of recreational beaches. Historically, detection of bacteria and viruses requires two different capture methods to detect both types of organisms in a given water sample. The purpose of this present study was to evaluate a newly devised dual layered filtration system, which was developed to simultaneously concentrate both viruses and bacteria in one step from marine waters. An apparatus was designed to accommodate two 90-mm diam., 0.45 microm pore size membranes in series, one on top of the other. The top polyvinylidene fluoride (PVDF) membrane was used to filter bacteria by physical straining while the bottom HA membrane retained viruses through adsorption. Results indicated that the dual layer filtration system recovered 83+/-14% of the test bacteria (Enterococcus fecalis) and 81+/-28% of the test virus (MS2 coliphage) on the top and bottom membranes, respectively. This research demonstrates the potential of using a dual layered filtration system for the simultaneous concentration of both bacteria and viruses on separate filters from recreational beach waters. This system is relatively simple to use, inexpensive, and has the potential to be suitable for routine monitoring. This study serves as a proof of concept for the technique. Additional experiments are needed to evaluate the system on a variety of different bacteria and viruses as well as on water with different physical and chemical parameters.

Faecal indicator bacteria enumeration in beach sand: a comparison study of extraction methods in medium to coarse sands.

AIMS: The absence of standardized methods for quantifying faecal indicator bacteria (FIB) in sand hinders comparison of results across studies. The purpose of the study was to compare methods for extraction of faecal bacteria from sands and recommend a standardized extraction technique. METHODS AND RESULTS: Twenty-two methods of extracting enterococci and Escherichia coli from sand were evaluated, including multiple permutations of hand shaking, mechanical shaking, blending, sonication, number of rinses, settling time, eluant-to-sand ratio, eluant composition, prefiltration and type of decantation. Tests were performed on sands from California, Florida and Lake Michigan. Most extraction parameters did not significantly affect bacterial enumeration. anova revealed significant effects of eluant composition and blending; with both sodium metaphosphate buffer and blending producing reduced counts. CONCLUSIONS: The simplest extraction method that produced the highest FIB recoveries consisted of 2 min of hand shaking in phosphate-buffered saline or deionized water, a 30-s settling time, one-rinse step and a 10 : 1 eluant volume to sand weight ratio. This result was consistent across the sand compositions tested in this study but could vary for other sand types. SIGNIFICANCE AND IMPACT OF THE STUDY: Method standardization will improve the understanding of how sands affect surface water quality.

Sequential concentration of bacteria and viruses from marine waters using a dual membrane system.

The ability to rapidly and effectively concentrate diverse microbes is an essential component for monitoring water quality at recreational beaches. The purpose of this study was to develop a 0.45 microm pore size dual membrane system, which can sequentially concentrate both viruses and bacteria. The top PVDF membrane was used to filter bacteria by physical straining while the bottom HA membrane retained viruses through adsorption. The recovery of this system was assessed using test organisms: enterococci and somatic coliphage. Volumes of 100 to 400 mL of unspiked and sewage-spiked beach water were filtered through both types of membranes. The PVDF membrane recovered statistically equivalent amounts of enterococci when compared to traditional membranes. All of the coliphage passed through the PVDF membrane, while 22% passed through the HA membrane. Increasing the volume from 100 to 400 mL did not significantly influence recoveries. Up to 35% of coliphage was eluted from the bottom membrane using beef extract solution. Rinsing bottom membranes with 0.5 mmol L(-1) H(2)S0(4) was found to deactivate somatic coliphage. This research demonstrates the potential of using a dual membrane adsorption system for the concentration of both bacteria and viruses from recreational beaches. A proposed bi-layer filtration system can be designed for simultaneous bacteria and virus filtration. Future experiments should focus on measurements utilizing additional bacteria and viruses.

Impacts of Hurricanes Katrina and Rita on the microbial landscape of the New Orleans area.

Floodwaters in New Orleans from Hurricanes Katrina and Rita were observed to contain high levels of fecal indicator bacteria and microbial pathogens, generating concern about long-term impacts of these floodwaters on the sediment and water quality of the New Orleans area and Lake Pontchartrain. We show here that fecal indicator microbe concentrations in offshore waters from Lake Pontchartrain returned to prehurricane concentrations within 2 months of the flooding induced by these hurricanes. Vibrio and Legionella species within the lake were more abundant in samples collected shortly after the floodwaters had receded compared with samples taken within the subsequent 3 months; no evidence of a long-term hurricane-induced algal bloom was observed. Giardia and Cryptosporidium were detected in canal waters. Elevated levels of fecal indicator bacteria observed in sediment could not be solely attributed to impacts from floodwaters, as both flooded and nonflooded areas exhibited elevated levels of fecal indicator bacteria. Evidence from measurements of Bifidobacterium and bacterial diversity analysis suggest that the fecal indicator bacteria observed in the sediment were from human fecal sources. Epidemiologic studies are highly recommended to evaluate the human health effects of the sediments deposited by the floodwaters.

Oceans and human health: Emerging public health risks in the marine environment.

There has been an increasing recognition of the inter-relationship between human health and the oceans. Traditionally, the focus of research and concern has been on the impact of human activities on the oceans, particularly through anthropogenic pollution and the exploitation of marine resources. More recently, there has been recognition of the potential direct impact of the oceans on human health, both detrimental and beneficial. Areas identified include: global change, harmful algal blooms (HABs), microbial and chemical contamination of marine waters and seafood, and marine models and natural products from the seas. It is hoped that through the recognition of the inter-dependence of the health of both humans and the oceans, efforts will be made to restore and preserve the oceans.

A pilot study of children's exposure to CCA-treated wood from playground equipment.

Arsenic from chromated copper arsenate (CCA)-treated wood, widely used in playgrounds and other outdoor equipment, can persist as surface residues on wood. This raises concerns about possible health risks associated with children playing on CCA-treated playgrounds. In a Pilot Study, 11 children (13-71 months) in homes with and without CCA-treated playgrounds were evaluated with post-exposure hand rinses and urine for total arsenic. Samples of wood, soil, and mulch, as well as synthetic wipes, were sampled for total arsenic. In non-CCA-treated playgrounds vs. CCA-treated playgrounds, respectively, wood arsenic was <2.0 mg/kg vs. mean arsenic 2370 mg/kg (range 1440-3270 mg/kg); soil arsenic was <3.0 mg/kg vs. mean arsenic of 19 mg/kg (range 4.0-42 mg/kg); mulch arsenic at one non-CCA-treated playground was 0.4 mg/kg vs. two CCA-treated playgrounds of 0.6 and 69 mg/kg. The arsenic removed using a synthetic wipe at non-CCA-treated playgrounds was <0.5 microg, while mean arsenic from CCA-treated wood was 117 microg (range 1.0-313). The arsenic mass from hand rinses for children who played at non-CCA-treated playgrounds was <0.2 microg, while mean arsenic mass was 0.6 microg (range <0.2-1.9) at CCA-treated playgrounds. Mean urinary total arsenic levels were 13.6 pg/ml (range 7.2-23.1 pg/ml) for all children evaluated, but there was no association between access to CCA-playgrounds and urinary arsenic levels. Arsenic speciation was not performed. This preliminary Pilot Study of CCA-treated wood playgrounds observed dislodgeable arsenic on 11 children's hands after brief periods of play exposure. Future efforts should increase the number of children and the play exposure periods, and incorporate speciation in order to discriminate between various sources of arsenic.

A Pilot Study of Microbial Contamination of Subtropical Recreational Waters.

Microbial water quality indicators are used to determine whether a water body is safe for recreational purposes. There have been concerns raised about the appropriate use of microbial indicators to regulate recreational uses of water bodies, in particular those located in tropical and sub-tropical environments. This prospective cohort pilot study evaluated the relationship between microbial water quality indicators and public health within two public beaches without known sewage discharge, but with historically high microbial levels for one beach, in subtropical Miami-Dade County (Florida). Monitoring was conducted in three phases: daily water monitoring, beach sand sampling, and spatially intense water sampling. An epidemiological questionnaire from a Los Angeles recreational beach-goer study was used to assess the self-reported swimming-related symptoms and exposures. There was no significant association between the number nor the type of reported symptoms and the different sampling months or beach sites, although persons who returned repeatedly to the beach were more likely to report symptoms. The number of indicator organisms correlated negatively with the frequency of symptoms reported by recreational beach goers. Results of the daily monitoring indicated that different indicators provided conflicting results concerning beach water quality.Larger epidemiologic studies with individual exposure monitoring are recommended to further evaluate these potentially important associations in subtropical recreational waters.

Sources of Escherichia coli in a coastal subtropical environment.

Sources of Escherichia coli in a coastal waterway located in Ft. Lauderdale, Fla., were evaluated. The study consisted of an extensive program of field measurements designed to capture spatial and temporal variations in E. coli concentrations as well as experiments conducted under laboratory-controlled conditions. E. coli from environmental samples was enumerated by using a defined substrate technology (Colilert-18). Field sampling tasks included sampling the length of the North Fork to identify the river reach contributing high E. coli levels, autosampler experiments at two locations, and spatially intense sampling efforts at hot spots. Laboratory experiments were designed to simulate tidal conditions within the riverbank soils. The results showed that E. coli entered the river in a large pulse during storm conditions. After the storm, E. coli levels returned to baseline levels and varied in a cyclical pattern which correlated with tidal cycles. The highest concentrations were observed during high tide, whereas the lowest were observed at low tide. This peculiar pattern of E. coli concentrations between storm events was caused by the growth of E. coli within riverbank soils which were subsequently washed in during high tide. Laboratory analysis of soil collected from the riverbanks showed increases of several orders of magnitude in soil E. coli concentrations. The ability of E. coli to multiply in the soil was found to be a function of soil moisture content, presumably due to the ability of E. coli to outcompete predators in relatively dry soil. The importance of soil moisture in regulating the multiplication of E. coli was found to be critical in tidally influenced areas due to periodic wetting and drying of soils in contact with water bodies. Given the potential for growth in such systems, E. coli concentrations can be artificially elevated above that expected from fecal impacts alone. Such results challenge the use of E. coli as a suitable indicator of water quality in tidally influenced areas located within tropical and subtropical environments.

Intestinal parasitic infections in human immunodeficiency virus (HIV)-positive and HIV-negative individuals in San Pedro Sula, Honduras.

Honduras has at least five-times more human immunodeficiency virus (HIV)-infected individuals than any other country in Central America. The relationship between HIV status and the presence of intestinal parasites in this part of the world is unknown. This study presents the results from a prospective, comparative study for the presence of parasites in 52 HIV-positive and 48 HIV-negative persons in San Pedro Sula, Honduras. Infection with HIV was determined by microagglutination and confirmed by Western blot analysis. Parasites were detected in stools using formalin-ether concentration, and Kinyoun and trichrome staining. Age, sex, and clinical state of HIV infection were recorded for each study participant. Our results indicate that Cryptosporidium parvum and Strongyloides stercoralis, which are intracellular or live in the mucosa, were found exclusively in persons infected with HIV. In comparison, the prevalence of the extracellular parasites Giardia lamblia, Ascaris lumbricoides, and Trichuris trichiura was significantly higher (P < 0.05) in persons who were HIV-negative. Trichuris worms are in contact with the gut epithelium and less so with the mucosa, whereas Strongyloides lives within the gut mucosa. It is possible that changes in the gut epithelium due to HIV infection do not affect the mucosa and therefore would not affect Strongyloides. We conclude that infection with HIV may selectively deter the establishment of certain intestinal parasites. This may be due to the fact that HIV-induced enteropathy does not favor the establishment of extracellular parasites. Intracellular and mucosal dwelling organisms, however, may benefit from pathologic changes and reduced local immune responses induced by the virus, which, in turn, may lead to higher prevalence among HIV-infected individuals.

Occurrence of Cryptosporidium oocysts and Giardia cysts in water supplies of San Pedro Sula, Honduras.

During June 1996, water supplies of the city of San Pedro Sula, Honduras, were sampled to obtain an assessment of Cryptosporidium oocyst and Giardia cyst concentrations. Each sample was concentrated and stained with an indirect immunofluorescent antibody, and parasites were counted through microscopic analysis. In three surface water supplies, Cryptosporidium oocyst concentrations ranged from 58 to 260 oocysts per 100 L, and Giardia cysts were present in concentrations ranging from 380 to 2100 cysts per 100 L. Unlike the surface water samples, groundwater had a higher concentration of Cryptosporidium oocysts (26/100 L) than Giardia cysts (6/100 L), suggesting that the groundwater aquifer protects the water supply more effectively from larger Giardia cysts. Cryptosporidium oocyst concentrations are within the typical range for surface water supplies in North America whereas Giardia cyst concentrations are elevated. Efforts should be made to protect raw water from sources of contamination.