Zika virus RNA persistence and recovery in water and wastewater: An approach for Zika virus surveillance in resource-constrained settings.

During the 2015-2016 Zika virus (ZIKV) epidemic in the Americas, serological cross-reactivity with other flaviviruses and relatively high costs of nucleic acid testing in the region hindered the capacity for widespread diagnostic testing. In such cases where individual testing is not feasible, wastewater monitoring approaches may offer a means of community-level public health surveillance. To inform such approaches, we characterized the persistence and recovery of ZIKV RNA in experiments where we spiked cultured ZIKV into surface water, wastewater, and a combination of both to examine the potential for detection in open sewers serving communities most affected by the ZIKV outbreak, such as those in Salvador, Bahia, Brazil. We used reverse transcription droplet digital PCR to quantify ZIKV RNA. In our persistence experiments, we found that the persistence of ZIKV RNA decreased with increasing temperature, significantly decreased in surface water versus wastewater, and significantly decreased when the initial concentration of virus was lowered by one order of magnitude. In our recovery experiments, we found higher percent recovery of ZIKV RNA in pellets versus supernatants from the same sample, higher recoveries in pellets using skimmed milk flocculation, lower recoveries of ZIKV RNA in surface water versus wastewater, and lower recoveries from a freeze thaw. We also analyzed samples collected from Salvador, Brazil during the ZIKV outbreak (2015-2016) that consisted of archived samples obtained from open sewers or environmental waters thought to be contaminated by sewage. Although we did not detect any ZIKV RNA in the archived Brazil samples, results from these persistence and recovery experiments serve to inform future wastewater monitoring efforts in open sewers, an understudied and important application of wastewater monitoring.

A comparison of SARS-CoV-2 wastewater concentration methods for environmental surveillance.

Wastewater1 surveillance of SARS-CoV-2 may be a useful supplement to clinical surveillance as it is shed in feces, there are many asymptomatic cases, and diagnostic testing can have capacity limitations and extended time to results. Although numerous studies have utilized wastewater surveillance for SARS-CoV-2, the methods used were developed and/or standardized for other pathogens. This study evaluates multiple methods for concentration and recovery of SARS-CoV-2 and seeded human coronavirus OC43 from municipal primary wastewater and/or sludge from the Greater Seattle Area (March-July 2020). Methods evaluated include the bag-mediated filtration system (BMFS), with and without Vertrel™ extraction, skimmed milk flocculation, with and without Vertrel™ extraction, polyethylene glycol (PEG) precipitation, ultrafiltration, and sludge extraction. Total RNA was extracted from wastewater concentrates and analyzed for SARS-CoV-2 and OC43 with RT-qPCR. Skimmed milk flocculation without Vertrel™ extraction performed consistently over time and between treatment plants in Seattle-area wastewater with the lowest average OC43 Cq value and smallest variability (24.3; 95% CI: 23.8-24.9), most frequent SARS-CoV-2 detection (48.8% of sampling events), and highest average OC43 percent recovery (9.1%; 95% CI: 6.2-11.9%). Skimmed milk flocculation is also beneficial because it is feasible in low-resource settings. While the BMFS had the highest average volume assayed of 11.9 mL (95% CI: 10.7-13.1 mL), the average OC43 percent recovery was low (0.7%; 95% CI: 0.4-1.0%). Ultrafiltration and PEG precipitation had low average OC43 percent recoveries of 1.0% (95% CI: 0.5-1.6%) and 3.2% (95% CI: 1.3-5.1%), respectively. The slopes and efficiency for the SARS-CoV-2 standard curves were not consistent over time, confirming the need to include a standard curve each run rather than using a single curve for multiple plates. Results suggest that the concentration and detection methods used must be validated for the specific water matrix using a recovery control to assess performance over time.

Review of Methods Suitable for Environmental Surveillance of Salmonella Typhi and Paratyphi.

Typhoid fever is an enteric disease caused by the pathogens Salmonella Typhi and Salmonella Paratyphi. Clinical surveillance networks are lacking in many affected areas, thus presenting a need to understand transmission and population prevalence. Environmental surveillance (ES) has been suggested as a potentially effective method in the absence of (or in supplement to) clinical surveillance. This review summarizes methods identified in the literature for sampling and detection of typhoidal Salmonella from environmental samples including drinking water, wastewater, irrigation water, and surface waters. Methods described use a trap or grab sampling approach combined with various selective culture and molecular methods. The level to which the performance of identified methods is characterized for ES in the literature is variable, thus arguing for the optimization and standardization of ES techniques.

Detection of potentially pathogenic enteric viruses in environmental samples from Kenya using the bag-mediated filtration system.

Enteric virus environmental surveillance via a highly sensitive method is critical, as many enteric viruses have low infectious doses and can persist in the environment for extended periods. This study determined the potential of the novel bag-mediated filtration system (BMFS) to recover human enteric viruses and pepper mild mottle virus (PMMoV) from wastewater and wastewater-impacted surface waters, examined PMMoV use as a fecal contamination indicator in Kenya, and identified potential BMFS process controls. From April 2015 to April 2016, BMFS samples were collected from seven sites in Kenya (n = 59). Enteroviruses and PMMoV were detected in 100% of samples, and human adenovirus, human astrovirus, hepatitis A virus, norovirus GI, norovirus GII, sapovirus, and human rotavirus were detected in the majority of samples. The consistent detection of enteroviruses and PMMoV suggests that these viruses could be used as indicators in similarly fecally contaminated sites and BMFS process controls. As contamination of surface water sources remains a global issue, enteric virus environmental surveillance is necessary. This study demonstrates an effective way to sample large volumes of wastewater and wastewater-impacted surface waters for the detection of multiple enteric viruses simultaneously.

Comparison of Risk Predicted by Multiple Norovirus Dose-Response Models and Implications for Quantitative Microbial Risk Assessment.

The application of quantitative microbial risk assessments (QMRAs) to understand and mitigate risks associated with norovirus is increasingly common as there is a high frequency of outbreaks worldwide. A key component of QMRA is the dose-response analysis, which is the mathematical characterization of the association between dose and outcome. For Norovirus, multiple dose-response models are available that assume either a disaggregated or an aggregated intake dose. This work reviewed the dose-response models currently used in QMRA, and compared predicted risks from waterborne exposures (recreational and drinking) using all available dose-response models. The results found that the majority of published QMRAs of norovirus use the 1 F1 hypergeometric dose-response model with α = 0.04, ß = 0.055. This dose-response model predicted relatively high risk estimates compared to other dose-response models for doses in the range of 1-1,000 genomic equivalent copies. The difference in predicted risk among dose-response models was largest for small doses, which has implications for drinking water QMRAs where the concentration of norovirus is low. Based on the review, a set of best practices was proposed to encourage the careful consideration and reporting of important assumptions in the selection and use of dose-response models in QMRA of norovirus. Finally, in the absence of one best norovirus dose-response model, multiple models should be used to provide a range of predicted outcomes for probability of infection.

Microbial safety and quality of fresh herbs from Los Angeles, Orange County and Seattle farmers' markets.

BACKGROUND: Farmers' markets have been growing in popularity in the United States, but the microbial quality and safety of the food sold at these markets is currently unknown. The purpose of this study was to assess the microbial safety and quality of fresh basil, parsley and cilantro sold at farmers' markets in the Los Angeles, Orange County and greater Seattle areas. RESULTS: A total of 133 samples (52 basil, 41 cilantro and 40 parsley) were collected from 13 different farmers' markets and tested for Salmonella and generic Escherichia coli. One sample (parsley) was confirmed positive for Salmonella and 24.1% of samples were positive for generic E. coli, with a range of 0.70-3.15 log CFU g(-1) . Among the herbs tested, basil showed the highest percentage of samples with generic E. coli (26.9%), followed by cilantro (24.4%) and then parsley (20.0%). For 12% of samples, the levels of generic E. coli exceeded guidelines established by the Public Health Laboratory Service for microbiological quality of ready-to-eat foods. CONCLUSION: Overall, this study indicates the presence of Salmonella and generic E. coli in fresh herbs sold at farmers' markets; however, additional studies are needed to determine the sources and extent of contamination.

Environment or host?: A case-control study of risk factors for Mycobacterium avium complex lung disease.

RATIONALE: Mycobacterium avium complex lung disease is an increasingly common and chronically debilitating problem. Several host traits have been suggested or confirmed as risk factors. Potential environmental and behavioral risk factors have also been proposed. Few have been evaluated in comparative studies. OBJECTIVES: To determine if aerosol-generating activities in the home and garden, features of the home water supply, or several pulmonary and immune-compromising conditions are associated with Mycobacterium avium complex lung disease. METHODS: Cases were recruited from academic medical centers and by informal referrals from nonuniversity practices in Washington and Oregon. Control subjects were recruited by random-digit dialing and matched to cases by age, sex, and partial telephone number. Associations were measured as odds ratios (OR) estimated using conditional logistic regression. MEASUREMENTS AND MAIN RESULTS: Known and potential risk factors were measured by in-home interview. Fifty-two matched pairs were studied. Six of 12 examined host traits were associated with disease, including history of chronic obstructive pulmonary disease (OR, 10; 95% confidence interval [CI], 1.2-80), pneumonia hospitalization (OR, 3.4; 95% CI, 1.1-11), and steroid use (OR, 8; 95% CI, 1.6-41). In contrast, 11 of the 14 aerosol-generating activities and all five features of home water supply studied bore little or no association with disease. CONCLUSIONS: Aerosol-generating activities seem not to be risk factors for Mycobacterium avium complex lung disease in HIV-negative adults, but prior lung disease and immune-suppressing drugs seem to be associated with susceptibility.

Methicillin-resistant Staphylococcus aureus from Northwest marine and freshwater recreational beaches.

The aim of the study was to determine the spatial distribution of methicillin-resistant Staphylococcus aureus (MRSA) at two marine and one freshwater recreational beaches in the Seattle area. Fifty-six marine water, 144 freshwater, and 96 sand samples were collected from June through August 2010. Isolates were biochemically verified as MRSA. Staphylococcal cassette chromosome mec (SCCmec) typing, multilocus sequence typing (MLST), pulse field gel electrophoresis and the presence of other antibiotic resistance genes were determined. Twenty-two freshwater (15.3%; n = 144), one dry sand (1.9%; n = 53), six wet sand (14%; n = 43), and two marine water samples (3.6%; n = 56) were MRSA positive. Of the 27 freshwater stream sites sampled multiple times, 37% of the sites were positive for MRSA and/or S. aureus ≥ 2 times. Twenty-one (67.7%) of 31 MRSA were SCCmec type IV, 15 (48.4%) of the isolates had MLST types not previously associated with humans, and 29 (93.5%) of the isolates carried other antibiotic resistance genes. This study is the first to report and characterize repeated MRSA-positive samples from freshwater drainages and creeks surrounding popular recreational beaches.

Survival of environmental and clinical strains of methicillin-resistant Staphylococcus aureus [MRSA] in marine and fresh waters.

Recent studies have found variable levels of methicillin-resistant Staphylococcus aureus [MRSA] in marine water from temperate and warmer climates suggesting that temperature may play a role in survival of MRSA in the environment. The aim of the study was to compare the survival of clinical and environmental MRSA and MSSA strains in fresh and marine water incubated at 13 °C and 20 °C over 14 days. Seven different MRSA strains and the MSSA ATCC 25923 were tested. Individual strains were diluted in sterile saline to a 0.5 McFarland standard (10(8) cfu/ml), serially diluted in duplicate to a final concentration of 10(5) cfu/ml in pooled filter-sterilized marine or fresh water and incubated at 13 °C or 20 °C in the dark. The results of this study found that temperature and salinity are important factors in MRSA and MSSA survival; the decay rate was ∼28% higher at 20 °C versus 13 °C and ∼34-44% higher in fresh water versus marine water. There was no statistical difference between environmental and clinical MRSA strain survival [P = 0.138]. The study found that MRSA/MSSA survival was significantly longer in marine water at 13 °C typical of the Pacific Northwest, which may have important implications for recreational beach visitors in colder climates.

Extraction of MS2 phage RNA from upper respiratory tract specimens by use of flat glass devices.

The isolation of pure nucleic acids from clinical samples is a crucial step in the molecular diagnosis of viral infections by nucleic acid testing (NAT). In this study, novel flat glass devices (cards) were demonstrated to support the rapid and efficient extraction of nucleic acids from upper respiratory tract specimens (nasal washes and swabs). The performance of the nucleic acid extraction cards was directly compared to an existing standardized and automated platform for viral extraction from these types of specimens. The flowthrough card method improved the speed of nucleic acid purification and accommodated larger sample volumes in extraction of bacteriophage MS2 RNA from the various specimen matrices. The dynamic range and estimated sensitivity of the card extraction method for reverse transcriptase quantitative real-time PCR (RT-qPCR)-based detection approximate those of the standardized magnetic glass bead extraction method used in this study.

An observational study on the effectiveness of point-of-use chlorination.

Although the efficacy of chlorine disinfection under controlled laboratory conditions is well known, the effectiveness of chlorine under field point-of-use (POU) conditions is still not clearly understood and may be impacted by a variety of factors. This study evaluated the effectiveness of POU chlorine disinfection in rural Ecuador under typical use conditions and compared this effectiveness with the efficacy in controlled laboratory conditions. While reductions of indicator organisms were slightly higher in households that used chlorination, no significant differences were seen between households employing POU chlorination and the households with no chlorination (1-1.5 log10 median reductions for chlorinating households and 0.31-0.55 log10 for nonchlorinating households, depending on the indicator organism). In contrast, significant reduction of all test organisms was found when simulating POU conditions in the laboratory. This study demonstrates that POU chlorination can be considerably less effective under actual field conditions than would be predicted based on its laboratory efficacy (3-5 log10 median reductions for chlorinated and 0-0.3 log10 for nonchlorinated samples). Human factors (including improper storage and chlorine dosing) and uncontrolled water quality effects are hypothesized to impact significantly the effectiveness of chlorine disinfection.

The coastal environment and human health: microbial indicators, pathogens, sentinels and reservoirs.

Innovative research relating oceans and human health is advancing our understanding of disease-causing organisms in coastal ecosystems. Novel techniques are elucidating the loading, transport and fate of pathogens in coastal ecosystems, and identifying sources of contamination. This research is facilitating improved risk assessments for seafood consumers and those who use the oceans for recreation. A number of challenges still remain and define future directions of research and public policy. Sample processing and molecular detection techniques need to be advanced to allow rapid and specific identification of microbes of public health concern from complex environmental samples. Water quality standards need to be updated to more accurately reflect health risks and to provide managers with improved tools for decision-making. Greater discrimination of virulent versus harmless microbes is needed to identify environmental reservoirs of pathogens and factors leading to human infections. Investigations must include examination of microbial community dynamics that may be important from a human health perspective. Further research is needed to evaluate the ecology of non-enteric water-transmitted diseases. Sentinels should also be established and monitored, providing early warning of dangers to ecosystem health. Taken together, this effort will provide more reliable information about public health risks associated with beaches and seafood consumption, and how human activities can affect their exposure to disease-causing organisms from the oceans.

Climatic and landscape correlates for potential West Nile virus mosquito vectors in the Seattle region.

Climatic and landscape patterns have been associated with both relative mosquito abundance and transmission of mosquito-borne illnesses in many parts of the world, especially warm and tropical climes. To determine if temperature, precipitation, or degree of urbanization were similarly important in the number of potential mosquito vectors for West Nile virus in the moderately temperate climate of western Washington, mosquitoes were collected using CDC carbon-dioxide/light traps set throughout the Seattle region during the summers of 2003 and 2004. The type and abundance of recovered species were compared to ecological correlates. Temperature and mosquito abundance were positively correlated, while precipitation was not strongly correlated with numbers of mosquitoes. Potential WNV mosquito vectors were most abundant in urban and suburban sites, including sites near communal roosts of American crows (Corvus brachyrhynchos). Exurban sites had the greatest vector species diversity, and Culex pipiens was the most abundant species throughout the region.