Continued selection on cryptic SARS-CoV-2 observed in Missouri wastewater.

Deep sequencing of wastewater to detect SARS-CoV-2 has been used during the COVID-19 pandemic to monitor viral variants as they appear and circulate in communities. SARS-CoV-2 lineages of an unknown source that have not been detected in clinical samples, referred to as cryptic lineages, are sometimes repeatedly detected from specific locations. We have continued to detect one such lineage previously seen in a Missouri site. This cryptic lineage has continued to evolve, indicating continued selective pressure similar to that observed in Omicron lineages.

Genetic diversity and evolutionary convergence of cryptic SARS- CoV-2 lineages detected via wastewater sequencing.

Wastewater-based epidemiology (WBE) is an effective way of tracking the appearance and spread of SARS-COV-2 lineages through communities. Beginning in early 2021, we implemented a targeted approach to amplify and sequence the receptor binding domain (RBD) of SARS-COV-2 to characterize viral lineages present in sewersheds. Over the course of 2021, we reproducibly detected multiple SARS-COV-2 RBD lineages that have never been observed in patient samples in 9 sewersheds located in 3 states in the USA. These cryptic lineages contained between 4 to 24 amino acid substitutions in the RBD and were observed intermittently in the sewersheds in which they were found for as long as 14 months. Many of the amino acid substitutions in these lineages occurred at residues also mutated in the Omicron variant of concern (VOC), often with the same substitutions. One of the sewersheds contained a lineage that appeared to be derived from the Alpha VOC, but the majority of the lineages appeared to be derived from pre-VOC SARS-COV-2 lineages. Specifically, several of the cryptic lineages from New York City appeared to be derived from a common ancestor that most likely diverged in early 2020. While the source of these cryptic lineages has not been resolved, it seems increasingly likely that they were derived from long-term patient infections or animal reservoirs. Our findings demonstrate that SARS-COV-2 genetic diversity is greater than what is commonly observed through routine SARS-CoV-2 surveillance. Wastewater sampling may more fully capture SARS-CoV-2 genetic diversity than patient sampling and could reveal new VOCs before they emerge in the wider human population.

Mercury, selenium, and fatty acids in the axial muscle of largemouth bass: evaluating the influence of seasonal and sexual changes in fish condition and reproductive status.

Largemouth bass (LMB, 265-475 mm) were collected to document whether changes in fish condition and reproductive status influenced the concentration of total mercury (Hg) and selenium (Se) in axial muscle by season and sex. The fatty acid (FA) composition of fish was also examined to describe seasonal and sexual differences and identify whether arachidonic acid (ARA) could be used as a biomarker of Hg toxicity. There was a trend for females to have lower (p < 0.062) Se concentrations than males. The concentration of Se for females during spring (mean ± SD, 686 ± 51 ng/g dw) was 15% lower than males (806 ± 67 ng/g dw). Lower Se concentrations in females than males continued through summer and fall. Concentration of Hg for females during spring (152 ± 39 ng/g ww) was also 59% lower than males (373 ± 303 ng/g ww), but the difference was not significant (p > 0.2). The percent of lipids was greatest in fall and winter (3%) and comprised primarily of omega-3 fatty acids (35 g/100 g lipid). Fish condition as measured by percent lipids and relative weight was negatively (p < 0.02) related to Hg concentration for females and males. Lipid content for both sexes was also positively (p < 0.05) related to the Se:Hg ratio. Relative weight was positively related to the Se:Hg ratio for females during all seasons (p = 0.014), but only during spring and summer for males (p < 0.007). A low Se:Hg value was associated with an elevation in ARA for both sexes and a reduced hepatosomatic index in males. Data suggested that females transferred muscle stores of Se and Hg to developing oocytes during spring. This study generates hypotheses regarding the physiological drivers of seasonal and sexual variability in Hg, Se, and FA in LMB that may be applicable to other species and have implications for fisheries health and management.

Cake Decorating Luster Dust Associated with Toxic Metal Poisonings - Rhode Island and Missouri, 2018-2019.

During 2018-2019, the Rhode Island Department of Health (RIDOH) and the Missouri Department of Health and Senior Services (DHSS) investigated cases of metal poisonings associated with commercially and home-prepared cakes decorated with products referred to as luster dust. Several types of glitters and dusts, broadly known as luster dust,* for use on prepared foods can be purchased online and in craft and bakery supply stores (1). Decorating foods with luster dust and similar products is a current trend, popularized on television programs, instructional videos, blogs, and in magazine articles.† Some luster dusts are specifically produced with edible ingredients that can be safely consumed. Companies that make edible luster dust are required by law to include a list of ingredients on the label (2). Luster dusts that are safe for consumption are typically marked "edible" on the label. Some luster dusts used as cake decorations are not edible or food grade; labeled as "nontoxic" or "for decorative purposes only," these luster dusts are intended to be removed before consumption (3). RIDOH (2018) and Missouri DHSS (2019), investigated heavy metal poisonings associated with commercially and home-prepared cakes decorated with luster dust after receiving reports of children (aged 1-11 years) who became ill after consuming birthday cake. Cases in Rhode Island were associated with copper ingestion, and the case in Missouri was associated with a child's elevated blood lead level. In Rhode Island, luster dust products that had been used in cake frosting were found to contain high levels of multiple metals.§ These events indicate that increased vigilance by public health departments and further guidance to consumers and bakeries are needed to prevent unintentional poisonings. Labeling indicating that a product is nontoxic does not imply that the product is safe for consumption. Explicit labeling indicating that nonedible products are not safe for human consumption is needed to prevent illness from inappropriate use of inedible products on foods. Educating consumers, commercial bakers, and public health professionals about potential hazards of items used in food preparation is essential to preventing illness and unintentional poisoning from toxic metals and other nonedible ingredients.

Physical, Chemical, and Biological Factors that Contribute to the Variability of Mercury Concentrations in Largemouth Bass Micropterus salmoides from Missouri Reservoirs.

Large-bodied predatory sportfish from Missouri reservoirs can contain elevated methylmercury concentrations that are of concern to the health of consumers. The concentration of total mercury (tHg) in the muscle (which > 95% is in the methylated-Hg form) of harvestable-sized largemouth bass (Micropterus salmoides; LMB) was examined to determine which factors contributed to the variability of tHg concentration in sportfish populations among Missouri reservoirs. Mean tHg concentrations in LMB from each reservoir were compared to physical and chemical characteristics of the reservoir and to biological attributes of each LMB population. Low concentrations of tHg (70-170 ng/g wet weight) in LMB from large reservoirs (surface area ≥ 35,680 acres) were likely related to the dilution of chemical Hg forms with water volume and depth. The highest tHg concentrations in LMB (268-542 ng/g) were from reservoirs with low particulate inorganic material (< 1.5 mg/L) and chlorophyll a concentrations (< 14.6 µg/L), and from LMB populations with a low proportion of large fish (proportional size distribution of LMB > 12 inches was < 33%). These relationships suggest that resource competition among LMB likely contributed to tHg bioaccumulation in reservoirs < 930 acres. Small reservoirs located in northern Missouri also may have greater methylation potential due to warmer water temperatures and anoxic conditions, but more data are needed to confirm these interactions. Fish consumption advisories for reservoirs with large surface area and volume could be reduced from one fish meal per month to one per week. To improve Missouri fisheries and protect consumers, management strategies to limit methylation and improve fish growth should be considered to reduce methylmercury bioaccumulation in small- and medium-sized reservoirs.

Mercury Spill Responses - Five States, 2012-2015.

Despite measures to educate the public about the dangers of elemental mercury, spills continue to occur in homes, schools, health care facilities, and other settings, endangering the public's health and requiring costly cleanup. Mercury is most efficiently absorbed by the lungs, and exposure to high levels of mercury vapor after a release can cause cough, sore throat, shortness of breath, nausea, vomiting, diarrhea, headaches, and visual disturbances (1). Children and fetuses are most susceptible to the adverse effects of mercury vapor exposure. Because their organ systems are still developing, children have increased respiratory rates, and they are closer to the ground where mercury vapors are most highly concentrated (2). To summarize key features of recent mercury spills and lessons learned, five state health departments involved in the cleanup (Iowa, Michigan, Missouri, North Carolina, and Wisconsin) compiled data from various sources on nonthermometer mercury spills from 2012 to 2015. The most common sites of contamination were residences, schools and school buses, health care facilities, and commercial and industrial facilities. Children aged <18 years were present in about one third of the spills, with approximately one in seven incidents resulting in symptoms consistent with acute mercury exposure. To protect the public's health after a mercury spill, it is important that local, state, and federal agencies communicate and coordinate effectively to ensure a quick response, and to minimize the spread of contamination. To reduce the number of mercury spills that occur in the United States, public health officials should increase awareness about exchange programs for mercury-containing items and educate school and health care workers about sources of mercury and how to dispose of them properly.

The different adsorption-degradation behaviors of SARS-CoV-2 by bioactive chemicals in wastewater: The suppression kinetics and their implications for wastewater-based epidemiology.

Wastewater-Based Epidemiology (WBE) is widely used to monitor the progression of SARS-CoV-2 pandemic. While there is a clear correlation between the number of COVID patients in a sewershed and the viral load in the wastewater, there is notable variability across different treatment plants. In particular, some facilities consistently exhibit higher viral content per diagnosed patient, implying a potential underestimation of the number of COVID patients, while others show a low viral load per diagnosed case, indicating potential attenuation of genetic material from the sewershed. In this study, we investigated the impact of nonylphenol ethoxylate (NPHE), linear alkylbenzene sulfonic acid (LABS), bisoctyl dimethyl ammonium chloride (BDAC), and didecyldimethylammonium chloride (DDAC), the surfactants that have been commonly used as detergents, emulsifiers, wetting agents on the stability of SARS-CoV-2 in wastewater. The results showed multiple and dynamic mechanisms, including degradation and desorption, can occur simultaneously during the interaction between SARS-CoV-2 and different chemicals depending on the physicochemical properties of each chemical. Through the elucidation of the dynamic interactions, the findings from this study could help the state health organizations and scientific community to optimize the SARS-CoV-2 wastewater-based epidemiology strategies.

Tracing the origin of SARS-CoV-2 omicron-like spike sequences detected in an urban sewershed: a targeted, longitudinal surveillance study of a cryptic wastewater lineage.

BACKGROUND: The origin of novel SARS-CoV-2 spike sequences found in wastewater, without corresponding detection in clinical specimens, remains unclear. We sought to determine the origin of one such cryptic wastewater lineage by tracking and characterising its persistence and genomic evolution over time. METHODS: We first detected a cryptic lineage, WI-CL-001, in municipal wastewater in Wisconsin, USA, in January, 2022. To determine the source of WI-CL-001, we systematically sampled wastewater from targeted sub-sewershed lines and maintenance holes using compositing autosamplers. Viral concentrations in wastewater samples over time were measured by RT digital PCR. In addition to using metagenomic 12s rRNA sequencing to determine the virus's host species, we also sequenced SARS-CoV-2 spike receptor binding domains, and, where possible, whole viral genomes to identify and characterise the evolution of this lineage. FINDINGS: We traced WI-CL-001 to its source at a single commercial building. There we detected the cryptic lineage at concentrations as high as 2·7 × 109 genome copies per L. The majority of 12s rRNA sequences detected in wastewater leaving the identified source building were human. Additionally, we generated over 100 viral receptor binding domain and whole-genome sequences from wastewater samples containing the cryptic lineage collected over the 13 consecutive months this virus was detectable (January, 2022, to January, 2023). These sequences contained a combination of fixed nucleotide substitutions characteristic of Pango lineage B.1.234, which circulated in humans in Wisconsin at low levels from October, 2020, to February, 2021. Despite this, mutations in the spike gene and elsewhere resembled those subsequently found in omicron variants. INTERPRETATION: We propose that prolonged detection of WI-CL-001 in wastewater indicates persistent shedding of SARS-CoV-2 from a single human initially infected by an ancestral B.1.234 virus. The accumulation of convergent omicron-like mutations in WI-CL-001's ancestral B.1.234 genome probably reflects persistent infection and extensive within-host evolution. People who shed cryptic lineages could be an important source of highly divergent viruses that sporadically emerge and spread. FUNDING: The Rockefeller Foundation, Wisconsin Department of Health Services, Centers for Disease Control and Prevention, National Institute on Drug Abuse, and the Center for Research on Influenza Pathogenesis and Transmission.

Microcystin accumulation in Sportfish from an agricultural reservoir differs among feeding guild, tissue type, and time of sampling.

Cyanobacterial blooms sometimes create secondary metabolites that can be transferred between trophic levels and accumulate in fish, but little is known about what time of year fish are most susceptible. Here, we examine microcystin in the muscle, liver, and kidney of bluegill and largemouth bass from an agricultural reservoir over 12 months. We identify which fish characteristics and water parameters best explain microcystin accumulation in fish tissues. Microcystin in bluegill was significantly higher than largemouth bass. In both species, microcystin was highest in livers (bluegill mean = 57.6 ng g - 1, largemouth bass mean = 71.8 ng g - 1 wet weight [ww]), then kidneys (bluegill mean = 27.1, largemouth bass mean = 22.7 ng g - 1 ww), followed by muscles (bluegill mean = 7.6, largemouth bass mean = 5.7 ng g - 1 ww). Adult bluegill feed on benthic macroinvertebrates and zooplankton, which may explain their higher microcystin concentrations compared to largemouth bass, which are primarily piscivorous. Harvest date emerged as the best predictor of microcystin in muscles and kidneys, with the highest concentrations occurring in April. Microcystin in water also emerged as a significant predictor, albeit much lower than harvest date, suggesting that low but persistent microcystin concentrations in water may result in accumulation of this cyanotoxin in fish. This study is the first to examine microcystin in fish from the North American Great Plains and one of only 5 studies that investigate microcystin in bluegill and largemouth bass. Additional investigation into the relationship between cyanobacteria and fish health is warranted, especially during spring when fish microcystin concentrations were highest.

Biomarkers selection for population normalization in SARS-CoV-2 wastewater-based epidemiology.

Wastewater-based epidemiology (WBE) has been one of the most cost-effective approaches to track the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) levels in the communities since the coronavirus disease 2019 (COVID-19) outbreak in 2020. Normalizing SARS-CoV-2 concentrations by the population biomarkers in wastewater is critical for interpreting the viral loads, comparing the epidemiological trends among the sewersheds, and identifying the vulnerable communities. In this study, five population biomarkers, pepper mild mottle virus (PMMoV), creatinine (CRE), 5-hydroxyindoleacetic acid (5-HIAA), caffeine (CAF) and its metabolite paraxanthine (PARA) were investigated and validated for their utility in normalizing the SARS-CoV-2 loads through two normalizing approaches using the data from 64 wastewater treatment plants (WWTPs) in Missouri. Their utility in assessing the real-time population contributing to the wastewater was also evaluated. The best performing candidate was further tested for its capacity for improving correlation between normalized SARS-CoV-2 loads and the clinical cases reported in the City of Columbia, Missouri, a university town with a constantly fluctuating population. Our results showed that, except CRE, the direct and indirect normalization approaches using biomarkers allow accounting for the changes in wastewater dilution and differences in relative human waste input over time regardless flow volume and population of the given WWTP. Among selected biomarkers, PARA is the most reliable population biomarker in determining the SARS-CoV-2 load per capita due to its high accuracy, low variability, and high temporal consistency to reflect the change in population dynamics and dilution in wastewater. It also demonstrated its excellent utility for real-time assessment of the population contributing to the wastewater. In addition, the viral loads normalized by the PARA-estimated population significantly improved the correlation (rho=0.5878, p < 0.05) between SARS-CoV-2 load per capita and case numbers per capita. This chemical biomarker complements the current normalization scheme recommended by CDC and helps us understand the size, distribution, and dynamics of local populations for forecasting the prevalence of SARS-CoV2 within each sewershed.

Defining biological and biophysical properties of SARS-CoV-2 genetic material in wastewater.

SARS-CoV-2 genetic material has been detected in raw wastewater around the world throughout the COVID-19 pandemic and has served as a useful tool for monitoring community levels of SARS-CoV-2 infections. SARS-CoV-2 genetic material is highly detectable in a patient's feces and the household wastewater for several days before and after a positive COVID-19 qPCR test from throat or sputum samples. Here, we characterize genetic material collected from raw wastewater samples and determine recovery efficiency during a concentration process. We find that pasteurization of raw wastewater samples did not reduce SARS-CoV-2 signal if RNA is extracted immediately after pasteurization. On the contrary, we find that signal decreased by approximately half when RNA was extracted 24-36 h post-pasteurization and ~90% when freeze-thawed prior to concentration. As a matrix control, we use an engineered enveloped RNA virus. Surprisingly, after concentration, the recovery of SARS-CoV-2 signal is consistently higher than the recovery of the control virus leading us to question the nature of the SARS-CoV-2 genetic material detected in wastewater. We see no significant difference in signal after different 24-hour temperature changes; however, treatment with detergent decreases signal ~100-fold. Furthermore, the density of the samples is comparable to enveloped retrovirus particles, yet, interestingly, when raw wastewater samples were used to inoculate cells, no cytopathic effects were seen indicating that wastewater samples do not contain infectious SARS-CoV-2. Together, this suggests that wastewater contains fully intact enveloped particles.

Biomarkers Selection for Population Normalization in SARS-CoV-2 Wastewater-based Epidemiology.

Wastewater-based epidemiology (WBE) has been one of the most cost-effective approaches to track the SARS-CoV-2 levels in the communities since the COVID-19 outbreak in 2020. Normalizing SARS-CoV-2 concentrations by the population biomarkers in wastewater can be critical for interpreting the viral loads, comparing the epidemiological trends among the sewersheds, and identifying the vulnerable communities. In this study, five population biomarkers, pepper mild mottle virus (pMMoV), creatinine (CRE), 5-hydroxyindoleacetic acid (5-HIAA), caffeine (CAF) and its metabolite paraxanthine (PARA) were investigated for their utility in normalizing the SARS-CoV-2 loads through developed direct and indirect approaches. Their utility in assessing the real-time population contributing to the wastewater was also evaluated. The best performed candidate was further tested for its capacity for improving correlation between normalized SARS-CoV-2 loads and the clinical cases reported in the City of Columbia, Missouri, a university town with a constantly fluctuated population. Our results showed that, except CRE, the direct and indirect normalization approaches using biomarkers allow accounting for the changes in wastewater dilution and differences in relative human waste input over time regardless flow volume and population at any given WWTP. Among selected biomarkers, PARA is the most reliable population biomarker in determining the SARS-CoV-2 load per capita due to its high accuracy, low variability, and high temporal consistency to reflect the change in population dynamics and dilution in wastewater. It also demonstrated its excellent utility for real-time assessment of the population contributing to the wastewater. In addition, the viral loads normalized by the PARA-estimated population significantly improved the correlation ( rho =0.5878, p <0.05) between SARS-CoV-2 load per capita and case numbers per capita. This chemical biomarker offers an excellent alternative to the currently CDC-recommended pMMoV genetic biomarker to help us understand the size, distribution, and dynamics of local populations for forecasting the prevalence of SARS-CoV2 within each sewershed. HIGHLIGHT bullet points: The paraxanthine (PARA), the metabolite of the caffeine, is a more reliable population biomarker in SARS-CoV-2 wastewater-based epidemiology studies than the currently recommended pMMoV genetic marker.SARS-CoV-2 load per capita could be directly normalized using the regression functions derived from correlation between paraxanthine and population without flowrate and population data.Normalizing SARS-CoV-2 levels with the chemical marker PARA significantly improved the correlation between viral loads per capita and case numbers per capita.The chemical marker PARA demonstrated its excellent utility for real-time assessment of the population contributing to the wastewater.

Genetic Diversity and Evolutionary Convergence of Cryptic SARS-CoV-2 Lineages Detected Via Wastewater Sequencing.

Wastewater-based epidemiology (WBE) is an effective way of tracking the appearance and spread of SARS-COV-2 lineages through communities. Beginning in early 2021, we implemented a targeted approach to amplify and sequence the receptor binding domain (RBD) of SARS-COV-2 to characterize viral lineages present in sewersheds. Over the course of 2021, we reproducibly detected multiple SARS-COV-2 RBD lineages that have never been observed in patient samples in 9 sewersheds located in 3 states in the USA. These cryptic lineages contained between 4 to 24 amino acid substitutions in the RBD and were observed intermittently in the sewersheds in which they were found for as long as 14 months. Many of the amino acid substitutions in these lineages occurred at residues also mutated in the Omicron variant of concern (VOC), often with the same substitution. One of the sewersheds contained a lineage that appeared to be derived from the Alpha VOC, but the majority of the lineages appeared to be derived from pre-VOC SARS-COV-2 lineages. Specifically, several of the cryptic lineages from New York City appeared to be derived from a common ancestor that most likely diverged in early 2020. While the source of these cryptic lineages has not been resolved, it seems increasingly likely that they were derived from immunocompromised patients or animal reservoirs. Our findings demonstrate that SARS-COV-2 genetic diversity is greater than what is commonly observed through routine SARS-CoV-2 surveillance. Wastewater sampling may more fully capture SARS-CoV-2 genetic diversity than patient sampling and could reveal new VOCs before they emerge in the wider human population. Author Summary: During the COVID-19 pandemic, wastewater-based epidemiology has become an effective public health tool. Because many infected individuals shed SARS-CoV-2 in feces, wastewater has been monitored to reveal infection trends in the sewersheds from which the samples were derived. Here we report novel SARS-CoV-2 lineages in wastewater samples obtained from 3 different states in the USA. These lineages appeared in specific sewersheds intermittently over periods of up to 14 months, but generally have not been detected beyond the sewersheds in which they were initially found. Many of these lineages may have diverged in early 2020. Although these lineages share considerable overlap with each other, they have never been observed in patients anywhere in the world. While the wastewater lineages have similarities with lineages observed in long-term infections of immunocompromised patients, animal reservoirs cannot be ruled out as a potential source.

Identification and quantification of bioactive compounds suppressing SARS-CoV-2 signals in wastewater-based epidemiology surveillance.

Recent SARS-CoV-2 wastewater-based epidemiology (WBE) surveillance have documented a positive correlation between the number of COVID-19 patients in a sewershed and the level of viral genetic material in the wastewater. Efforts have been made to use the wastewater SARS-CoV-2 viral load to predict the infected population within each sewershed using a multivariable regression approach. However, reported clear and sustained variability in SARS-CoV-2 viral load among treatment facilities receiving industrial wastewater have made clinical prediction challenging. Several classes of molecules released by regional industries and manufacturing facilities, particularly the food processing industry, can significantly suppress the SARS-CoV-2 signals in wastewater by breaking down the lipid-bilayer of the membranes. Therefore, a systematic ranking process in conjugation with metabolomic analysis was developed to identify the wastewater treatment facilities exhibiting SARS-CoV-2 suppression and identify and quantify the chemicals suppressing the SARS-COV-2 signals. By ranking the viral load per diagnosed case among the sewersheds, we successfully identified the wastewater treatment facilities in Missouri, USA that exhibit SARS-CoV-2 suppression (significantly lower than 5 × 1011 gene copies/reported case) and determined their suppression rates. Through both untargeted global chemical profiling and targeted analysis of wastewater samples, 40 compounds were identified as candidates of SARS-CoV-2 signal suppressors. Among these compounds, 14 had higher concentrations in wastewater treatment facilities that exhibited SARS-CoV-2 signal suppression compared to the unsuppressed control facilities. Stepwise regression analyses indicated that 4-nonylphenol, palmitelaidic acid, sodium oleate, and polyethylene glycol dioleate are positively correlated with SARS-CoV-2 signal suppression rates. Suppression activities were further confirmed by incubation studies, and the suppression kinetics for each bioactive compound were determined. According to the results of these experiments, bioactive molecules in wastewater can significantly reduce the stability of SARS-CoV-2 genetic marker signals. Based on the concentrations of these chemical suppressors, a correction factor could be developed to achieve more reliable and unbiased surveillance results for wastewater treatment facilities that receive wastewater from similar industries.

Monitoring SARS-CoV-2 Populations in Wastewater by Amplicon Sequencing and Using the Novel Program SAM Refiner.

Sequencing Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) from wastewater has become a useful tool in monitoring the spread of viral variants. Approaches to this task have been varied, relying on differing sequencing methods and computational analyses. We used a novel computation workflow based on amplicon sequencing of SARS-CoV-2 spike domains in order to track viral populations in wastewater. As part of this workflow, we developed a program, SAM Refiner, that has a variety of outputs, including novel variant reporting as well as functions designed to remove polymerase chain reaction (PCR) generated chimeric sequences. With these methods, we were able to track viral population dynamics over time. We report here on the emergence of two variants of concern, B.1.1.7 (Alpha) and P.1 (Gamma), and their displacement of the D614G B.1 variant in a Missouri sewershed.