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Mark D Sobsey - One of the best experts on this subject based on the ideXlab platform.
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occurrence of male specific and somatic Coliphages and relationship with rainfall in privately owned wells from peri urban and rural households
Water research X, 2021Co-Authors: Megan A Stallard, Mark D Sobsey, Riley E Mulhern, Emily Greenwood, Taylor Franklin, Lawrence S Engel, Michael Fisher, Hania Zanib, Rachel T Noble, Jill R StewartAbstract:Abstract Privately-owned drinking water wells serving fewer than 25 people (private wells) are prevalent and understudied across most of the US. Private wells primarily serve rural households located outside of municipal drinking water and sewerage service coverage areas. These wells are not regulated by United States Environmental Protection Agency (EPA) under the Safe Drinking Water Act, are not regularly monitored by any public agency or utility, and generally do not undergo disinfection treatment. Coliphages are a group of viruses that infect coliform bacteria and are useful viral surrogates for fecal contamination in water systems in much the same way that fecal indicator bacteria (FIB), such as E. coli and to a lesser extent total coliforms, are used to quantify fecal contamination. Coliphages are approved by the EPA for regulatory monitoring in groundwater wells in the USA, but are not routinely used for this purpose. The present study characterizes the occurrence of male-specific and somatic Coliphages, along with FIB, in private wells (n = 122) across two different counties in North Carolina. While occurrences of E. coli were rare and frequency of total coliform was generally low (~20%), male-specific and somatic Coliphages were detectable in 66% and 54% of samples, respectively. Concentrations of male-specific Coliphages were higher than somatics at each county and on a monthly basis. Rainfall appears to be partly influencing higher coliphage concentrations in December, January and February. This research underscores the need for increased surveillance in private wells and consideration of using Coliphages in order to better characterize occurrence of fecal contamination at the time of sampling, especially during rainier months.
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e coli cb390 an alternative e coli host for simultaneous detection of somatic and f coliphage viruses in reclaimed and other waters
Journal of Virological Methods, 2017Co-Authors: Emily S Bailey, Matthew Price, Lisa M Casanova, Mark D SobseyAbstract:Somatic and F+ Coliphages have been identified and validated as virus indicators of fecal contamination in ground water by US EPA and more recently they are being considered for use in managing both marine and fresh recreational water and wastewater discharges. Studies documenting their usefulness as viral indicator in reclaimed water sources in the USA are limited. However, simultaneous detection of both somatic and F+ Coliphages on a single E. coli host is preferred over their separate analysis because both are abundant in wastewater, they may respond differently to wastewater reclamation treatment processes, and separate analysis for each group in separate host bacteria adds complexity and cost. In this study, a new total coliphage host (E. coli CB390, CECT9198) was evaluated for its ability to detect somatic, F+ Coliphages, and total Coliphages by US EPA Methods 1601 and 1602. No statistical difference was found in the detection Coliphages in spiked phosphate buffered saline samples or in natural waters; additionally, no statistical difference was found between the detection of total Coliphages by Methods 1601 and 1602.
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comparison of methods for the detection of Coliphages in recreational water at two california united states beaches
Journal of Virological Methods, 2012Co-Authors: Roberto A Rodriguez, Jill R Stewart, David C. Love, Julianne Tajuba, Jacqueline Knee, Jerold W Dickerson, Laura F Webster, Mark D SobseyAbstract:a b s t r a c t Methods for detection of two fecal indicator viruses, F+ and somatic Coliphages, were evaluated for appli- cation to recreational marine water. Marine water samples were collected during the summer of 2007 in Southern California, United States from transects along Avalon Beach (n = 186 samples) and Doheny Beach (n = 101 samples). Coliphage detection methods included EPA method 1601 - two-step enrichment (ENR), EPA method 1602 - single agar layer (SAL), and variations of ENR. Variations included comparison of two incubation times (overnight and 5-h incubation) and two final detection steps (lysis zone assay and a rapid latex agglutination assay). A greater number of samples were positive for somatic and F+ col- iphages by ENR than by SAL (p < 0.01). The standard ENR with overnight incubation and detection by lysis zone assay was the most sensitive method for the detection of F+ and somatic Coliphages from marine water, although the method takes up to three days to obtain results. A rapid 5-h enrichment version of ENR also performed well, with more positive samples than SAL, and could be performed in roughly 24 h. Latex agglutination-based detection methods require the least amount of time to perform, although the sensitivity was less than lysis zone-based detection methods. Rapid culture-based enrichment of col- iphages in marine water may be possible by further optimizing culture-based methods for saline water conditions to generate higher viral titers than currently available, as well as increasing the sensitivity of latex agglutination detection methods.
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Survival of prototype strains of somatic coliphage families in environmental waters and when exposed to UV low-pressure monochromatic radiation or heat.
Water Research, 2011Co-Authors: Mark D SobseyAbstract:Abstract The potential use of specific somatic coliphage taxonomic groups as viral indicators based on their persistence and prevalence in water was investigated. Representative type strains of the 4 major somatic coliphage taxonomic groups were seeded into reagent water and an ambient surface water source of drinking water and the survival of the added phages was measured over 90 days at temperatures of 23–25 and 4 °C. Microviridae (type strain PhiX174), Siphoviridae (type strain Lambda), and Myoviridae (type strain T4) viruses were the most persistent in water at the temperatures tested. The Microviridae (type strain PhiX174) and the Siphoviridae (type strain Lambda) were the most resistant viruses to UV radiation and the Myoviridae (type strain T4) and the Microviridae (type strain PhiX174) were the most resistant viruses to heat. Based on their greater persistence in water over time and their relative resistance to heat and/or UV radiation, the Myoviridae (type strain T4), the Microviridae (type strain PhiX174), and the Siphoviridae (type strain Lambda) were the preferred candidate somatic Coliphages as fecal indicator viruses in water, with the Microviridae (type strain PhiX174) the most resistant to these conditions overall.
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evaluation of rt pcr and reverse line blot hybridization for detection and genotyping f rna Coliphages from estuarine waters and molluscan shellfish
Journal of Applied Microbiology, 2008Co-Authors: David C. Love, Jan Vinje, S M Khalil, J Murphy, Greg L Lovelace, Mark D SobseyAbstract:Aims: To evaluate a PCR-based detection and typing method for faecal indicator viruses (F+ RNA Coliphages) in water and shellfish, and apply the method for better understanding of the ecology and microbial source tracking potential of these viruses. Methods and Results: Water and shellfish samples were collected over 3 years at nine estuaries in the East, West and Gulf Coasts of the USA, providing 1033 F+ RNA coliphage isolates. F+ RNA coliphage genotyping rates by reverse transcriptase-PCR – reverse line blot (RLB) hybridization ranged from 94·7% to 100% among estuaries, and were not significantly different in oysters, clams, mussels or water (P = 0·8427). Twenty samples negative by RLB were nucleotide sequenced for confirmation, and to refine RLB probes. More F+ RNA Coliphages were genotyped from colder water than warmer waters, while the water salinity did not affect F+ RNA coliphage levels. Conclusions: RT-PCR–RLB was a robust method for detecting and genotyping F+ RNA Coliphages from diverse coastal areas, which provided new information on the ecology of F+ RNA Coliphages. Significance and Impact of the Study: This performance-validated F+ RNA coliphage method can be used for faecal indicator monitoring and microbial source tracking, to protect recreational bathers and shellfish consumers from exposure to pathogenic virus and their disease risks.
Juan Jofre - One of the best experts on this subject based on the ideXlab platform.
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Fast and easy methods for the detection of Coliphages
Journal of microbiological methods, 2020Co-Authors: Anicet R. Blanch, Maite Muniesa, Francisco Lucena, Juan JofreAbstract:Somatic and F-specific Coliphages are gaining ground as indicators of fecal/viral pollution. Guidelines and regulations worldwide for monitoring water, biosolids and food are including them as parameters to assess quality and treatment efficiency. Robust methods to detect and quantify both groups of phages in water samples have been launched by agencies such as the International Standardization Organization (ISO) and the USA Environmental Protection Agency (USEPA). Although these methods have proved readily implementable in routine microbiology laboratories, faster and more user-friendly protocols will be highly welcome if coliphage detection becomes routine in water quality analysis. We here provide an overview of new approaches seeking to facilitate the detection of infectious Coliphages included in guidelines and regulations. The improvements achieved suggest that streamlined kits able to provide results in a few hours at very reasonable costs will become available in the near future. The potential of molecular procedures and methods based on microelectronic sensors is also briefly discussed.
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New approach for the simultaneous detection of somatic Coliphages and F-specific RNA Coliphages as indicators of fecal pollution
The Science of the total environment, 2018Co-Authors: Daniel Toribio-avedillo, Juan Jofre, Julia Martín-díaz, Anicet R. Blanch, Maite MuniesaAbstract:Two groups of Coliphages have been recently included in different water management policies as indicators of viral fecal pollution in water and food: somatic Coliphages, which infect E. coli through cell wall receptors, and F-specific RNA Coliphages, which infect through the F-pili. Somatic Coliphages are more abundant in fecally contaminated waters, except reclaimed waters, those disinfected by UV irradiation, and some groundwater samples that show a higher level of F-specific Coliphages. Somatic Coliphages are morphologically similar to DNA enteric viruses while F-specific Coliphages are similar to RNA viruses such as norovirus and hepatitis A viruses, which are the viral pathogens of concern in sewage. The use of strains sensitive to both types of phages has been proposed for total coliphage enumeration, thereby avoiding double analysis. The standardized methods available for coliphage detection are robust and cost-effective, but the introduction of ready-to-use methods would facilitate routine implementation in laboratories. The fastest available tool for somatic coliphage enumeration is the recently developed Bluephage, which uses a modified β-glucuronide-overexpressing E. coli strain unable to take up the glucuronide substrate. The overexpressed enzyme accumulates inside the bacterial cells until released by phage-induced cell lysis, whereupon it encounters its substrate and the medium changes from yellow to blue. The present method uses E. coli strain CB12, sensitive to somatic Coliphages and F-specific Coliphages due to the expression of the F-pili. The Bluephage approach incorporating CB12 detects both types of Coliphages in a time range of 1:30 to 4:00 h, as assayed with Coliphages from raw sewage, river water, sludge and mussels. This strategy can be applied to obtain qualitative and quantitative results and is applicable to microplates as well as to large sample volumes (100 ml). Moreover it can provide monitoring of water bodies at real time, as for example for ambient recreational beach monitoring.
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use of non linear mixed effects modelling and regression analysis to predict the number of somatic Coliphages by plaque enumeration after 3 hours of incubation
Journal of Water and Health, 2017Co-Authors: J Mendez, Juan Jofre, Antonio Monleongetino, F LucenaAbstract:The present study aimed to establish the kinetics of the appearance of coliphage plaques using the double agar layer titration technique to evaluate the feasibility of using traditional coliphage plaque forming unit (PFU) enumeration as a rapid quantification method. Repeated measurements of the appearance of plaques of Coliphages titrated according to ISO 10705-2 at different times were analysed using non-linear mixed-effects regression to determine the most suitable model of their appearance kinetics. Although this model is adequate, to simplify its applicability two linear models were developed to predict the numbers of Coliphages reliably, using the PFU counts as determined by the ISO after only 3 hours of incubation. One linear model, when the number of plaques detected was between 4 and 26 PFU after 3 hours, had a linear fit of: (1.48 × Counts 3 h + 1.97); and the other, values >26 PFU, had a fit of (1.18 × Counts 3 h + 2.95). If the number of plaques detected was
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simultaneous detection of somatic and f specific Coliphages in different settings by escherichia coli strain cb390
Fems Microbiology Letters, 2016Co-Authors: Miriam Agullobarcelo, F Lucena, Belen Galofre, Lluis Sala, Cristina Garciaaljaro, Juan JofreAbstract:Bacteriophages are increasingly being used as water quality indicators. Two groups of phages infecting Escherichia coli, somatic and F-specific Coliphages, are being considered as indicators of fecal and viral contamination for several types of water around the world. However, some uncertainties remain regarding which Coliphages to assess. Recently, E. coli strain CB390 has been reported to be suitable for simultaneous detection of both groups, which seems to be more informative than determining only one of the groups. Here, a significant number of samples from different settings, mostly those where F-specific phages have been reported to outnumber somatic Coliphages, are analyzed for somatic Coliphages, F-specific RNA phages by standardized methods and Coliphages detected by host strain CB390. The results presented here confirm that the numbers of phages counted using CB390 are equivalent to the sum of the somatic and F-specific Coliphages counted independently in all settings. Hence the usefulness of this strain for simultaneous detection of somatic and F-specific Coliphages is confirmed. Also, sets of data on the presence of Coliphages in reclaimed and groundwater are reported.
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Is the replication of somatic Coliphages in water environments significant
Journal of applied microbiology, 2008Co-Authors: Juan JofreAbstract:Somatic Coliphages are amid several groups of bacteriophages that have been suggested as indicators in water quality assessment. One of the limitations frequently endorsed to somatic Coliphages as indicators is that they can replicate in the water environment. This review intends to evaluate the significance of this potential replication. In view of: the threshold densities of somatic Coliphages and host bacteria needed for productive infection to occur, the densities of both host cells supporting somatic Coliphages replication and these phages in water environments, and the poor contribution of lysogenic induction to the free somatic coliphage numbers in water, it can be concluded that replication of somatic Coliphages in waters is very unlikely. Consequently, the contribution of replication in the environment of somatic Coliphages is expected to have a non-noticeable influence on the numbers of somatic Coliphages detected in water environments. Thus, the replication in the environment should not be argued as a limitation to the use of somatic Coliphages as indicators.
David C Smith - One of the best experts on this subject based on the ideXlab platform.
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multiplex quantitative real time reverse transcriptase pcr for f specific rna Coliphages a method for use in microbial source tracking
Applied and Environmental Microbiology, 2007Co-Authors: Marek Kirs, David C SmithAbstract:It is well documented that microbial contamination of coastal waters poses a significant risk to human health through recreational exposure and consumption of shellfish. Identifying the source of microbial contamination (microbial source tracking) plays a dominant role in enabling effective management and remediation strategies. One method used to determine the source of the contamination is quantification of the ratio of the four subgroups of F-specific RNA Coliphages (family Leviviridae) in impacted water samples. Because of typically low concentrations in the environment, enrichment assays are performed prior to detection, even though differential replication rates have been reported. These assays are also compromised by differential loss of phage infectivity among subgroups after release into the environment, thus obscuring the initial ratio. Here, a culture-independent multiplex real-time reverse transcriptase-PCR (RT-PCR) protocol for the simultaneous quantification of all four subgroups of F-specific RNA Coliphages using novel primer sets and molecular beacons is presented. This assay is extremely sensitive, achieving detection with as few as 10 copies of isolated coliphage RNA, and is linear for a minimum of six orders of magnitude. During survival experiments, the real-time RT-PCR technique was able to quantify Coliphages in seawater when culture-based double agar layer assay failed. While infectivity was lost at different rates at the subgroup level, decay constants in seawater, calculated using the real-time RT-PCR estimates, did not vary among subgroups. The accurate determination of the in situ concentration of F-specific RNA Coliphages using this method will facilitate more effective remediation strategies for impacted environments. Microbial contamination of lakes, rivers, estuarine, and coastal waters poses a major risk to human health through consumption of organisms that inhabit these environments as well as through recreational exposure. Identifying the source of microbial contamination (microbial source tracking) plays a dominant role in the types of actual risk and enabling effective management and remediation strategies. In their current form, monitoring programs and identification protocols for deter
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Quantitative real-time reverse transcriptionpolymerase chain reaction analysis of drug metabolizing and cytoprotective genes in psoriasis and regulation by ultraviolet radiation
2003Co-Authors: Marek Kirs, David C SmithAbstract:It is well documented that microbial contamination of coastal waters poses a significant risk to human health through recreational exposure and consumption of shellfish. Identifying the source of microbial con-tamination (microbial source tracking) plays a dominant role in enabling effective management and remedia-tion strategies. One method used to determine the source of the contamination is quantification of the ratio of the four subgroups of F-specific RNA Coliphages (family Leviviridae) in impacted water samples. Because of typically low concentrations in the environment, enrichment assays are performed prior to detection, even though differential replication rates have been reported. These assays are also compromised by differential loss of phage infectivity among subgroups after release into the environment, thus obscuring the initial ratio. Here, a culture-independent multiplex real-time reverse transcriptase-PCR (RT-PCR) protocol for the simultaneous quantification of all four subgroups of F-specific RNA Coliphages using novel primer sets and molecular beacons is presented. This assay is extremely sensitive, achieving detection with as few as 10 copies of isolated coliphage RNA, and is linear for a minimum of six orders of magnitude. During survival experiments, the real-time RT-PCR technique was able to quantify Coliphages in seawater when culture-based double agar layer assay failed. While infectivity was lost at different rates at the subgroup level, decay constants in seawater, calculated using the real-time RT-PCR estimates, did not vary among subgroups. The accurate determinatio
Marek Kirs - One of the best experts on this subject based on the ideXlab platform.
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multiplex quantitative real time reverse transcriptase pcr for f specific rna Coliphages a method for use in microbial source tracking
Applied and Environmental Microbiology, 2007Co-Authors: Marek Kirs, David C SmithAbstract:It is well documented that microbial contamination of coastal waters poses a significant risk to human health through recreational exposure and consumption of shellfish. Identifying the source of microbial contamination (microbial source tracking) plays a dominant role in enabling effective management and remediation strategies. One method used to determine the source of the contamination is quantification of the ratio of the four subgroups of F-specific RNA Coliphages (family Leviviridae) in impacted water samples. Because of typically low concentrations in the environment, enrichment assays are performed prior to detection, even though differential replication rates have been reported. These assays are also compromised by differential loss of phage infectivity among subgroups after release into the environment, thus obscuring the initial ratio. Here, a culture-independent multiplex real-time reverse transcriptase-PCR (RT-PCR) protocol for the simultaneous quantification of all four subgroups of F-specific RNA Coliphages using novel primer sets and molecular beacons is presented. This assay is extremely sensitive, achieving detection with as few as 10 copies of isolated coliphage RNA, and is linear for a minimum of six orders of magnitude. During survival experiments, the real-time RT-PCR technique was able to quantify Coliphages in seawater when culture-based double agar layer assay failed. While infectivity was lost at different rates at the subgroup level, decay constants in seawater, calculated using the real-time RT-PCR estimates, did not vary among subgroups. The accurate determination of the in situ concentration of F-specific RNA Coliphages using this method will facilitate more effective remediation strategies for impacted environments. Microbial contamination of lakes, rivers, estuarine, and coastal waters poses a major risk to human health through consumption of organisms that inhabit these environments as well as through recreational exposure. Identifying the source of microbial contamination (microbial source tracking) plays a dominant role in the types of actual risk and enabling effective management and remediation strategies. In their current form, monitoring programs and identification protocols for deter
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Quantitative real-time reverse transcriptionpolymerase chain reaction analysis of drug metabolizing and cytoprotective genes in psoriasis and regulation by ultraviolet radiation
2003Co-Authors: Marek Kirs, David C SmithAbstract:It is well documented that microbial contamination of coastal waters poses a significant risk to human health through recreational exposure and consumption of shellfish. Identifying the source of microbial con-tamination (microbial source tracking) plays a dominant role in enabling effective management and remedia-tion strategies. One method used to determine the source of the contamination is quantification of the ratio of the four subgroups of F-specific RNA Coliphages (family Leviviridae) in impacted water samples. Because of typically low concentrations in the environment, enrichment assays are performed prior to detection, even though differential replication rates have been reported. These assays are also compromised by differential loss of phage infectivity among subgroups after release into the environment, thus obscuring the initial ratio. Here, a culture-independent multiplex real-time reverse transcriptase-PCR (RT-PCR) protocol for the simultaneous quantification of all four subgroups of F-specific RNA Coliphages using novel primer sets and molecular beacons is presented. This assay is extremely sensitive, achieving detection with as few as 10 copies of isolated coliphage RNA, and is linear for a minimum of six orders of magnitude. During survival experiments, the real-time RT-PCR technique was able to quantify Coliphages in seawater when culture-based double agar layer assay failed. While infectivity was lost at different rates at the subgroup level, decay constants in seawater, calculated using the real-time RT-PCR estimates, did not vary among subgroups. The accurate determinatio
Sunny C. Jiang - One of the best experts on this subject based on the ideXlab platform.
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smartphone based in gel loop mediated isothermal amplification glamp system enables rapid coliphage ms2 quantification in environmental waters
Environmental Science & Technology, 2018Co-Authors: Xiao Huang, Sunny C. Jiang, Xingyu Lin, Katharina Urmann, Xing Xie, Michael R HoffmannAbstract:Model Coliphages (e.g., ΦX174, MS2, and PRD1) have been widely used as surrogates to study the fate and transport of pathogenic viruses in the environment and during wastewater treatment. Two groups of Coliphages (F-specific and somatic) are being explored as indicators of viral fecal pollution in ambient water. However, the detection and quantification of Coliphages still largely rely on time-consuming culture-based plaque assays. In this study, we developed an in-gel loop-mediated isothermal amplification (gLAMP) system enabling coliphage MS2 quantification within 30 min using standard laboratory devices. Viral particles (MS2) were immobilized with LAMP reagents in polyethylene glycol hydrogel, and then viral RNAs were amplified through a LAMP reaction. Due to the restriction effect of the hydrogel matrix, one viral particle would only produce one amplicon dot. Therefore, the sample virus concentrations can be determined based on the number of fluorescent amplicon dots using a smartphone for imaging. Th...
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Ecology of Coliphages in southern California coastal waters.
Journal of applied microbiology, 2010Co-Authors: V.c. Reyes, Sunny C. JiangAbstract:Aims: This study aims to investigate the ecology of Coliphages, an important microbial pollution indicator. Specifically, our experiments address (i) the ability of environmental Escherichia coli (E. coli) to serve as hosts for coliphage replication, and (ii) the temporal and spatial distribution of Coliphages in coastal waters. Methods and Results: Water samples from three locations in California’s Newport Bay watershed were tested for the presence of Coliphages every 2 weeks for an entire year. A total of nine E. coli strains isolated from various sources served as hosts for coliphage detection. Coliphage occurrence was significantly different between freshwater, estuarine and coastal locations and correlated with water temperature, salinity and rainfall in the watershed. The Coliphages isolated on the environmental hosts had a broad host-range relative to the Coliphages isolated on an E. coli strain from sewage and a US EPA recommended strain for coliphage detection. Conclusions: Coliphage occurrence was related to the temperature, rainfall and salinity within the bay. The adaptation to a broad host-range may enable the proliferation of Coliphages in the aquatic environment. Significance and Impact of the Study: Understanding the seasonal variation of phages is useful for establishing a background level of coliphage presence in coastal waters. The broad host-range of Coliphages isolated on the environmental E. coli host calls for investigation of coliphage replication in the aquatic
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human adenoviruses and Coliphages in urban runoff impacted coastal waters of southern california
Applied and Environmental Microbiology, 2001Co-Authors: Sunny C. Jiang, Rachel T Noble, Weiping ChuAbstract:A nested-PCR method was used to detect the occurrence of human adenovirus in coastal waters of Southern California. Twenty- to forty-liter water samples were collected from 12 beach locations from Malibu to the border of Mexico between February and March 1999. All sampling sites were located at mouths of major rivers and creeks. Two ultrafiltration concentration methods, tangential flow filtration (TFF) and vortex flow filtration (VFF), were compared using six environmental samples. Human adenoviruses were detected in 4 of the 12 samples tested after nucleic acid extraction of VFF concentrates. The most probable number of adenoviral genomes ranged from 880 to 7,500 per liter of water. Coliphages were detected at all sites, with the concentration varying from 5.3 to 3332 PFU/liter of water. F-specific Coliphages were found at 5 of the 12 sites, with the concentration ranging from 5.5 to 300 PFU/liter. The presence of human adenovirus was not significantly correlated with the concentration of coliphage (r = 0.32) but was significantly correlated (r = 0.99) with F-specific coliphage. The bacterial indicators (total coliforms, fecal coliforms, and enterococci) were found to exceed California recreational water quality daily limits at 5 of the 12 sites. However, this excess of bacterial indicators did not correlate with the presence of human adenoviruses in coastal waters. The results of this study call for both a reevaluation of our current recreational water quality standards to reflect the viral quality of recreational waters and monitoring of recreational waters for human viruses on a regular basis.