Persistence of fecal indicator bacteria and associated genetic markers from wastewater treatment plant effluents in freshwater microcosms.

Limited information exists on the environmental persistence of genetic markers for fecal indicator bacteria (FIB) in treated wastewaters. Here, the decay rate constants of culturable cells and genetic markers for four diverse groups of FIBs, such as enterococci, Clostridium, Escherichia coli, and Bacteroides, were investigated in freshwater microcosms seeded with disinfected and non-disinfected secondary-treated wastewaters. Decay rate constants of genetic markers and culturable cells varied significantly among the different FIB groups. Water temperatures (winter vs. fall/spring/summer) significantly affected the decay of all genetic marker and cell types; however, genetic marker decay were not found to be significantly different in disinfected (chlorination/ultraviolet) and non-disinfected wastewater-seeded microcosms or, for example, lake- and river-receiving waters. No evidence was seen that decay rate constants of FIB genetic markers from treated wastewater were substantially different from those observed in similar, previously reported microcosm studies using raw sewage. Unexpected relationships between decay rate constants of genetic markers and culturable cells of Bacteroides were observed. Results suggest that decay rate constants of FIB genetic markers determined from other studies may be applicable to treated wastewaters. Results of this study should be informative for ongoing efforts to determine the persistence of FIB genetic markers relative to surviving pathogens after wastewater treatment.

Rapid Salivary IgG Antibody Screening for Hepatitis A.

Hepatitis A virus (HAV) is a common infection that is transmitted through the fecal-oral route, shed in the stool of infected individuals, and spread either by direct contact or by ingesting contaminated food or water. Each year, approximately 1.4 million acute cases are reported globally with a major risk factor for exposure being low household socioeconomic status. Recent trends show a decrease in anti-HAV antibodies in the general population, with concomitant increases in the numbers of HAV outbreaks. In line with a recreational water study, this effort aims to assess the prevalence of salivary IgG antibodies against HAV and subsequent incident infections (or immunoconversions) in visitors to a tropical beach impacted by a publicly owned treatment works (POTW). We applied a multiplex immunoassay to serially collected saliva samples gathered from study participants who recreated at Boquerón Beach, Puerto Rico. Analysis of assay results revealed an immunoprevalence rate of 16.17% for HAV with 1.43% of the cohort immunoconverting to HAV. Among those who immunoconverted, 10% reported chronic gastrointestinal symptoms and none experienced diarrhea. Tests on water samples indicated good water quality with low levels of fecal indicator bacteria; however, the collection and analysis of saliva samples afforded the ability to detect HAV infections in beachgoers. This rapid assay serves as a cost-effective tool for examining exposure to environmental pathogens and can provide critical information to policy makers, water quality experts, and risk assessment professionals seeking to improve and protect recreational water and public health.

Assessment of two volumetrically different concentration approaches to improve sensitivities for SARS-CoV-2 detection during wastewater monitoring.

Wastewater monitoring for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), the virus responsible for the global coronavirus disease 2019 (COVID-19) pandemic, has highlighted the need for methodologies capable of assessing viral prevalence during periods of low population infection. To address this need, two volumetrically different, methodologically similar concentration approaches were compared for their abilities to detect viral nucleic acid and infectious SARS-CoV-2 signal from primary influent samples. For Method 1, 2 L of SARS-CoV-2 seeded wastewater was evaluated using a dead-end hollow fiber ultrafilter (D-HFUF) for primary concentration, followed by the CP Select™ for secondary concentration. For Method 2, 100 mL of SARS-CoV-2 seeded wastewater was evaluated using the CP Select™ procedure. Following D-HFUF concentration (Method 1), significantly lower levels of infectious SARS-CoV-2 were lost (P value range: 0.0398-0.0027) compared to viral gene copy (GC) levels detected by the US Centers for Disease Control (CDC) N1 and N2 reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) assays. Subsamples at different steps in the concentration process were also taken to better characterize the losses of SARS-CoV-2 during the concentration process. During the centrifugation step (prior to CP Select™ concentration), significantly higher losses (P value range: 0.0003 to <0.0001) occurred for SARS-CoV-2 GC levels compared to infectious virus for Method 1, while between the methods, significantly higher infectious viral losses were observed for Method 2 (P = 0.0002). When analyzing overall recovery of endogenous SARS-CoV-2 in wastewater samples, application of Method 1 improved assay sensitivities (P = <0.0001) compared with Method 2; this was especially evident during periods of lower COVID-19 case rates within the sewershed. This study describes a method which can successfully concentrate infectious SARS-CoV-2 and viral RNA from wastewater. Moreover, we demonstrated that large volume wastewater concentration provides additional sensitivity needed to improve SARS-CoV-2 detection, especially during low levels of community disease prevalence.

Interlaboratory comparison of real-time PCR protocols for quantification of general fecal indicator bacteria.

The application of quantitative real-time PCR (qPCR) technologies for the rapid identification of fecal bacteria in environmental waters is being considered for use as a national water quality metric in the United States. The transition from research tool to a standardized protocol requires information on the reproducibility and sources of variation associated with qPCR methodology across laboratories. This study examines interlaboratory variability in the measurement of enterococci and Bacteroidales concentrations from standardized, spiked, and environmental sources of DNA using the Entero1a and GenBac3 qPCR methods, respectively. Comparisons are based on data generated from eight different research facilities. Special attention was placed on the influence of the DNA isolation step and effect of simplex and multiplex amplification approaches on interlaboratory variability. Results suggest that a crude lysate is sufficient for DNA isolation unless environmental samples contain substances that can inhibit qPCR amplification. No appreciable difference was observed between simplex and multiplex amplification approaches. Overall, interlaboratory variability levels remained low (<10% coefficient of variation) regardless of qPCR protocol.

Bacterial and viral fecal indicator predictive modeling at three Great Lakes recreational beach sites.

Coliphage are viruses that infect Escherichia coli (E. coli) and may indicate the presence of enteric viral pathogens in recreational waters. There is an increasing interest in using these viruses for water quality monitoring and forecasting; however, the ability to use statistical models to predict the concentrations of coliphage, as often done for cultured fecal indicator bacteria (FIB) such as enterococci and E. coli, has not been widely assessed. The same can be said for FIB genetic markers measured using quantitative polymerase chain reaction (qPCR) methods. Here we institute least-angle regression (LARS) modeling of previously published concentrations of cultured FIB (E. coli, enterococci) and coliphage (F+, somatic), along with newly reported genetic concentrations measured via qPCR for E. coli, enterococci, and general Bacteroidales. We develop site-specific models from measures taken at three beach sites on the Great Lakes (Grant Park, South Milwaukee, WI; Edgewater Beach, Cleveland, OH; Washington Park, Michigan City, IN) to investigate the efficacy of a statistical predictive modeling approach. Microbial indicator concentrations were measured in composite water samples collected five days per week over a beach season (∼15 weeks). Model predictive performance (cross-validated standardized root mean squared error of prediction [SRMSEP] and R2PRED) were examined for seven microbial indicators (using log10 concentrations) and water/beach parameters collected concurrently with water samples. Highest predictive performance was seen for qPCR-based enterococci and Bacteroidales models, with F+ coliphage consistently yielding poor performing models. Influential covariates varied by microbial indicator and site. Antecedent rainfall, bird abundance, wave height, and wind speed/direction were most influential across all models. Findings suggest that some fecal indicators may be more suitable for water quality forecasting than others at Great Lakes beaches.

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.

Large-scale comparison of E. coli levels determined by culture and a qPCR method (EPA Draft Method C) in Michigan towards the implementation of rapid, multi-site beach testing.

Fecal pollution remains a challenge for water quality managers at Great Lakes and inland recreational beaches. The fecal indicator of choice at these beaches is typically Escherichia coli (E. coli), determined by culture-based methods that require over 18 h to obtain results. Researchers at the United States Environmental Protection Agency (EPA) have developed a rapid E. coli qPCR methodology (EPA Draft Method C) that can provide same-day results for improving public health protection with demonstrated sensitivity, specificity, and data acceptance criteria. However, limited information is currently available to compare the occurrence of E. coli determined by cultivation and by EPA Draft Method C (Method C). This study provides a large-scale data collection effort to compare the occurrence of E. coli determined by these alternative methods at more than 100 Michigan recreational beach and other sites using the complete set of quantitative data pairings and selected subsets of the data and sites meeting various eligibility requirements. Simple linear regression analyses of composite (pooled) data indicated a correlation between results of the E. coli monitoring approaches for each of the multi-site datasets as evidenced by Pearson R-squared values ranging from 0.452 to 0.641. Theoretical Method C threshold values, expressed as mean log10 target gene copies per reaction, that corresponded to an established E. coli culture method water quality standard of 300 MPN or CFU /100 mL varied only from 1.817 to 1.908 for the different datasets using this model. Different modeling and derivation approaches that incorporated within and between-site variability in the estimates also gave Method C threshold values in this range but only when relatively well-correlated datasets were used to minimize the error. A hypothetical exercise to evaluate the frequency of water impairments based on theoretical qPCR thresholds corresponding to the E. coli water quality standard for culture methods suggested that the methods may provide the same beach notification outcomes over 90% of the time with Method C results differing from culture method results that indicated acceptable and unacceptable water quality at overall rates of 1.9% and 6.6%, respectively. Results from this study provide useful information about the relationships between E. coli determined by culture and qPCR methods across many diverse freshwater sites and should facilitate efforts to implement qPCR-based E. coli detection for rapid recreational water quality monitoring on a large scale in the State of Michigan.

Salivary Antibodies against Multiple Environmental Pathogens Found in Individuals Recreating at an Iowa Beach.

Detecting environmental exposures and mitigating their impacts are growing global public health challenges. Antibody tests show great promise and have emerged as fundamental tools for large-scale exposure studies. Here, we apply, demonstrate and validate the utility of a salivary antibody multiplex immunoassay in measuring antibody prevalence and immunoconversions to six pathogens commonly found in the environment. The study aimed to assess waterborne infections in consenting beachgoers recreating at an Iowa riverine beach by measuring immunoglobulin G (IgG) antibodies against select pathogens in serially collected saliva samples. Results showed that nearly 80% of beachgoers had prior exposures to at least one of the targeted pathogens at the beginning of the study. Most of these exposures were to norovirus GI.1 (59.41%), norovirus GII.4 (58.79%) and Toxoplasma gondii (22.80%) and over half (56.28%) of beachgoers had evidence of previous exposure to multiple pathogens. Of individuals who returned samples for each collection period, 6.11% immunoconverted to one or more pathogens, largely to noroviruses (GI.1: 3.82% and GII.4: 2.29%) and T. gondii (1.53%). Outcomes of this effort illustrate that the multiplex immunoassay presented here serves as an effective tool for evaluating health risks by providing valuable information on the occurrence of known and emerging pathogens in population surveillance studies.

Performance evaluation of a dead-end hollowfiber ultrafiltration method for enumeration of somatic and F+ coliphage from recreational waters.

Dead-end hollow fiber ultrafiltration combined with a single agar layer assay (D-HFUF-SAL) has potential use in the assessment of sanitary quality of recreational waters through enumeration of coliphage counts as measures of fecal contamination. However, information on applicability across a broad range of sites and water types is limited. Here, we tested the performance of D-HFUF-SAL on 49 marine and freshwater samples. Effect of method used to titer the spiking suspension (SAL versus double agar layer [DAL]) on percent recovery was also evaluated. Average somatic coliphage recovery (72 % ± 27) was significantly higher (p < 0.0001) compared to F+ (53 % ± 19). This was more pronounced for marine (p ≤ 0.0001) compared to freshwaters (p = 0.0134). Neither method affected somatic coliphage, but DAL (28 % ± 12) significantly (p < 0.0001) underestimated F + coliphage recoveries compared to SAL (53 % ± 19). Overall, results indicate that, while D-HFUF-SAL performed well over a wide variety of water types, F + coliphage recoveries were significantly reduced for marine waters suggesting that some components unique to this habitat may interfere with the assay performance. More importantly, our findings indicate that choice of spike titer method merits careful consideration since it may under-estimate method percent recovery.

Development of a large volume concentration method for recovery of coronavirus from wastewater.

Levels of severe acute respiratory coronavirus type 2 (SARS CoV 2) RNA in wastewater could act as an effective means to monitor coronavirus disease 2019 (COVID-19) within communities. However, current methods used to detect SARS CoV 2 RNA in wastewater are limited in their ability to process sufficient volumes of source material, inhibiting our ability to assess viral load. Typically, viruses are concentrated from large liquid volumes using two stage concentration, primary and secondary. Here, we evaluated a dead-end hollow fiber ultrafilter (D-HFUF) for primary concentration, followed by the CP Select™ for secondary concentration from 2 L volumes of primary treated wastewater. Various amendments to each concentration procedure were investigated to optimally recover seeded OC43 (betacoronavirus) from wastewater. During primary concentration, the D-HFUF recovered 69 ± 18% (n = 29) of spiked OC43 from 2 L of wastewater. For secondary concentration, the CP Select™ system using the Wastewater Application settings was capable of processing 100 mL volumes of primary filter eluates in <25 min. A hand-driven syringe elution proved to be significantly superior (p = 0.0299) to the CP Select™ elution for recovering OC43 from filter eluates, 48 ± 2% compared to 31 ± 3%, respectively. For the complete method (primary and secondary concentration combined), the D-HFUF and CP select/syringe elution achieved overall 22 ± 4% recovery of spiked OC43 through (n = 8) replicate filters. Given the lack of available standardized methodology confounded by the inherent limitations of relying on viral RNA for wastewater surveillance of SARS CoV 2, it is important to acknowledge these challenges when interpreting this data to estimate community infection rates. However, the development of methods that can substantially increase sample volumes will likely allow for reporting of quantifiable viral data for wastewater surveillance, equipping public health officials with information necessary to better estimate community infection rates.

A Gallus gallus Model for Determining Infectivity of Zoonotic Campylobacter.

To better understand public health implications of waterfowl as reservoirs for zoonotic sources of Campylobacter in recreational waters, we developed a Gallus gallus (chick) model of infection to assess the pathogenicity of environmental isolates of Campylobacter. This method involved exposure of 1-day-old chicks through ingestion of water, the natural route of infection. Viable Campylobacter from laboratory-infected animals were monitored by using a modified non-invasive sampling of fresh chick excreta followed by a passive polycarbonate-filter migration culture assay. The method was used to evaluate the infectivities of three laboratory strains of Campylobacter spp. (Campylobacter coli, Campylobacter jejuni, and Campylobacter lari), three clinical isolates of C. jejuni, and four environmental Campylobacter spp. isolated from California gulls (Larus californicus). The results revealed that chicks were successfully infected with all laboratory and clinical isolates of Campylobacter spp. through ingestion of Campylobacter-spiked water, with infection rates ranging from <10 to >90% in a dose-dependent manner. More importantly, exposure of chicks with Campylobacter spp. isolated from Gallus gallus excreta also resulted in successful establishment of infection (≤90%). Each monitored Campylobacter spp. contained ≥7.5 × 104 CFU⋅g-1 of feces 7 days post-exposure. These results suggest that a G. gallus model can be used to assess infectivity of Campylobacter isolates, including gull and human clinical isolates. Use of an avian animal model can be applied to assess the importance of birds, such as the G. gallus, as potential contributors of waterborne-associated outbreaks of campylobacteriosis.

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.

Visitors to a Tropical Marine Beach Show Evidence of Immunoconversions to Multiple Waterborne Pathogens.

Determining infections from environmental exposures, particularly from waterborne pathogens is a challenging proposition. The study design must be rigorous and account for numerous factors including study population selection, sample collection, storage, and processing, as well as data processing and analysis. These challenges are magnified when it is suspected that individuals may potentially be infected by multiple pathogens at the same time. Previous work demonstrated the effectiveness of a salivary antibody multiplex immunoassay in detecting the prevalence of immunoglobulin G (IgG) antibodies to multiple waterborne pathogens and helped identify asymptomatic norovirus infections in visitors to Boquerón Beach, Puerto Rico. In this study, we applied the immunoassay to three serially collected samples from study participants within the same population to assess immunoconversions (incident infections) to six waterborne pathogens: Helicobacter pylori, Campylobacter jejuni, Toxoplasma gondii, hepatitis A virus, and noroviruses GI. I and GII.4. Further, we examined the impact of sampling on the detection of immunoconversions by comparing the traditional immunoconversion definition based on two samples to criteria developed to capture trends in three sequential samples collected from study participants. The expansion to three samples makes it possible to capture the IgG antibody responses within the survey population to more accurately assess the frequency of immunoconversions to target pathogens. Based on the criteria developed, results showed that when only two samples from each participant were used in the analysis, 25.9% of the beachgoers immunoconverted to at least one pathogen; however, the addition of the third sample reduced immunoconversions to 6.5%. Of these incident infections, the highest levels were to noroviruses followed by T. gondii. Moreover, many individuals displayed evidence of immunoconversions to multiple pathogens. This study suggests that detection of simultaneous infections is possible, with far reaching consequences for the population. The results may lead to further studies to understand the complex interactions that occur within the body as the immune system attempts to ward off these infections. Such an approach is critical to our understanding of medically important synergistic or antagonistic interactions and may provide valuable and critical information to public health officials, water treatment personnel, and environmental managers.

Evaluation of multiple laboratory performance and variability in analysis of recreational freshwaters by a rapid Escherichia coli qPCR method (Draft Method C).

There is interest in the application of rapid quantitative polymerase chain reaction (qPCR) methods for recreational freshwater quality monitoring of the fecal indicator bacteria Escherichia coli (E. coli). In this study we determined the performance of 21 laboratories in meeting proposed, standardized data quality acceptance (QA) criteria and the variability of target gene copy estimates from these laboratories in analyses of 18 shared surface water samples by a draft qPCR method developed by the U.S. Environmental Protection Agency (EPA) for E. coli. The participating laboratories ranged from academic and government laboratories with more extensive qPCR experience to "new" water quality and public health laboratories with relatively little previous experience in most cases. Failures to meet QA criteria for the method were observed in 24% of the total 376 test sample analyses. Of these failures, 39% came from two of the "new" laboratories. Likely factors contributing to QA failures included deviations in recommended procedures for the storage and preparation of reference and control materials. A master standard curve calibration model was also found to give lower overall variability in log10 target gene copy estimates than the delta-delta Ct (ΔΔCt) calibration model used in previous EPA qPCR methods. However, differences between the mean estimates from the two models were not significant and variability between laboratories was the greatest contributor to overall method variability in either case. Study findings demonstrate the technical feasibility of multiple laboratories implementing this or other qPCR water quality monitoring methods with similar data quality acceptance criteria but suggest that additional practice and/or assistance may be valuable, even for some more generally experienced qPCR laboratories. Special attention should be placed on providing and following explicit guidance on the preparation, storage and handling of reference and control materials.

Standardized data quality acceptance criteria for a rapid Escherichia coli qPCR method (Draft Method C) for water quality monitoring at recreational beaches.

There is growing interest in the application of rapid quantitative polymerase chain reaction (qPCR) and other PCR-based methods for recreational water quality monitoring and management programs. This interest has strengthened given the publication of U.S. Environmental Protection Agency (EPA)-validated qPCR methods for enterococci fecal indicator bacteria (FIB) and has extended to similar methods for Escherichia coli (E. coli) FIB. Implementation of qPCR-based methods in monitoring programs can be facilitated by confidence in the quality of the data produced by these methods. Data quality can be determined through the establishment of a series of specifications that should reflect good laboratory practice. Ideally, these specifications will also account for the typical variability of data coming from multiple users of the method. This study developed proposed standardized data quality acceptance criteria that were established for important calibration model parameters and/or controls from a new qPCR method for E. coli (EPA Draft Method C) based upon data that was generated by 21 laboratories. Each laboratory followed a standardized protocol utilizing the same prescribed reagents and reference and control materials. After removal of outliers, statistical modeling based on a hierarchical Bayesian method was used to establish metrics for assay standard curve slope, intercept and lower limit of quantification that included between-laboratory, replicate testing within laboratory, and random error variability. A nested analysis of variance (ANOVA) was used to establish metrics for calibrator/positive control, negative control, and replicate sample analysis data. These data acceptance criteria should help those who may evaluate the technical quality of future findings from the method, as well as those who might use the method in the future. Furthermore, these benchmarks and the approaches described for determining them may be helpful to method users seeking to establish comparable laboratory-specific criteria if changes in the reference and/or control materials must be made.

The development of point-of-use water filters as sampling devices in bioforensics: extent of microbial sorption and elution.

The foundational idea for this project is that household faucet-mounted water filters may be used as bioforensic sampling devices to detect the extent of a potential bioagent release in domestic water supplies. An optimized eluent solution was determined experimentally by quantifying recoveries of microorganisms from point-of-use (POU) drinking water filters. The optimized extraction protocol was then used in mock bioagent release experiments to determine the feasibility of POU filters as bioforensic sampling devices. Bacillus atrophaeus spores, Escherichia coli and PP7 virus were exposed to filters and the number of attached organisms was determined by enumerating the unattached organisms on selective agar media. Subsequently, the filters were eluted and the percent of extracted organisms was determined based on the number of attached organisms. Two popular brands of carbon block filters retained 92%-99% of representative virus, spore and vegetative bacteria. In back-flush elutions of single filters, the most efficient eluent was identified as a combination of 1% peptone and 1% Tween-80, and extraction recovered 25.4% (+/-17.5%) of attached E. coli, 20.4% (+/-3.6%) of B. atrophaeus spores, and 9.4% (+/-5.2%) of PP7 virions (+/- standard deviations). In bioagent release studies in which filters were challenged with 100 agents mL(-1), greater than 99% of the spores were retained by the filters, and the percent of attached spores that were recovered ranged from 10.4% at day 0 to 4.3% five days after the release event (averaged from five separate experiments). In contrast, E. coli, Salmonella typhimurium and PP7 virus were rapidly inactivated in the chlorinated tap water, indicating their improbable survival in chlorinated water supplies. It is therefore concluded that household water filters can be used as microbial sampling devices for bioforensic applications in the event of a bioagent release in domestic drinking water supplies.

Comparison of somatic and F+ coliphage enumeration methods with large volume surface water samples.

Coliphages are alternative fecal indicators that may be suitable surrogates for viral pathogens, but majority of standard detection methods utilize insufficient volumes for routine detection in environmental waters. We compared three somatic and F+ coliphage methods based on a paired measurement from 1 L samples collected from the Great Lakes (n = 74). Methods include: 1) dead-end hollow fiber ultrafilter with single agar layer (D-HFUF-SAL); 2) modified SAL (M-SAL); and 3) direct membrane filtration (DMF) technique. Overall, D-HFUF-SAL outperformed other methods as it yielded the lowest frequency of non-detects [(ND); 10.8%] and the highest average concentrations of recovered coliphage for positive samples (2.51 ± 1.02 [standard deviation, SD] log10 plaque forming unit/liter (PFU/L) and 0.79 ± 0.71 (SD) log10 PFU/L for somatic and F+, respectively). M-SAL yielded 29.7% ND and average concentrations of 2.26 ± 1.15 (SD) log10 PFU/L (somatic) and 0.59 ± 0.82 (SD) log10 PFU/L (F+). DMF performance was inferior to D-HFUF-SAL and M-SAL methods (ND of 65.6%; average somatic coliphage concentration 1.52 ± 1.32 [SD] log10 PFU/L, no F+ detected), indicating this procedure is unsuitable for 1 L surface water sample volumes. This study represents an important step toward the use of a coliphage method for recreational water quality criteria purposes.

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.

Incidence of somatic and F+ coliphage in Great Lake Basin recreational waters.

There is a growing interest for the use of coliphage as an alternative indicator to assess fecal pollution in recreational waters. Coliphage are a group of viruses that infect Escherichia coli and are considered as potential surrogates to infer the likely presence of enteric viral pathogens. We report the use of a dead-end hollow fiber ultrafiltration single agar layer method to enumerate F+ and somatic coliphage from surface waters collected from three Great Lake areas. At each location, three sites (two beaches; one river) were sampled five days a week over the 2015 beach season (n = 609 total samples). In addition, culturable E. coli and enterococci concentrations, as well as 16 water quality and recreational area parameters were assessed such as rainfall, turbidity, dissolved oxygen, pH, and ultra violet absorbance. Overall, somatic coliphage levels ranged from non-detectable to 4.39 log10 plaque forming units per liter and were consistently higher compared to F+ (non-detectable to 3.15 log10 PFU/L), regardless of sampling site. Coliphage concentrations weakly correlated with cultivated fecal indicator bacteria levels (E. coli and enterococci) at 75% of beach sites tested in study (r = 0.28 to 0.40). In addition, ultraviolet light absorption and water temperature were closely associated with coliphage concentrations, but not fecal indicator bacteria levels suggesting different persistence trends in Great Lake waters between indicator types (bacteria versus virus). Finally, implications for coliphage water quality management and future research directions are discussed.

Immunoprevalence to Six Waterborne Pathogens in Beachgoers at Boquerón Beach, Puerto Rico: Application of a Microsphere-Based Salivary Antibody Multiplex Immunoassay.

Waterborne infectious diseases are a major public health concern worldwide. Few methods have been established that are capable of measuring human exposure to multiple waterborne pathogens simultaneously using non-invasive samples such as saliva. Most current methods measure exposure to only one pathogen at a time, require large volumes of individual samples collected using invasive procedures, and are very labor intensive. In this article, we applied a multiplex bead-based immunoassay capable of measuring IgG antibody responses to six waterborne pathogens simultaneously in human saliva to estimate immunoprevalence in beachgoers at Boquerón Beach, Puerto Rico. Further, we present approaches for determining cutoff points to assess immunoprevalence to the pathogens in the assay. For the six pathogens studied, our results show that IgG antibodies against antigens from noroviruses GI.I and GII.4 were more prevalent (60 and 51.6%, respectively) than Helicobacter pylori (21.4%), hepatitis A virus (20.2%), Campylobacter jejuni (8.7%), and Toxoplasma gondii (8%) in the saliva of the study participants. The salivary antibody multiplex immunoassay can be used to examine immunoprevalence of specific pathogens in human populations.