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Norovirus GII wastewater monitoring for epidemiological surveillance [1]

['Michelle L. Ammerman', 'Department Of Civil', 'Environmental Engineering', 'University Of Michigan', 'Ann Arbor', 'Michigan', 'United States Of America', 'Shreya Mullapudi', 'Department Of Epidemiology', 'School Of Public Health']

Date: 2024-06 

Abstract While the Centers for Disease Control and Prevention coordinates several outbreak and clinical surveillance systems for norovirus, norovirus is strongly under-reported due to individuals not seeking care or not being tested. As a result, norovirus surveillance using case reports and syndromic detection often lags rather than leads outbreaks. Digital epidemiology sources such as search term data may be more immediate, but can be affected by behavior and media patterns. Wastewater monitoring can potentially provide a comprehensive and consistent data stream that can help to triangulate across these different data sets. To assess the timeliness of norovirus wastewater testing compared with syndromic, outbreak and search term trend data for norovirus, we quantified human norovirus GII in composite influent samples from 5 wastewater treatment plants (WWTPs) using reverse transcription-digital droplet PCR and correlated wastewater levels to syndromic, outbreak, and search term trend data. Wastewater human norovirus (HuNoV) GII RNA levels were comparable across all WWTPs after fecal content normalization using Pepper mild mottle virus (PMMoV). HuNoV GII wastewater values typically led syndromic, outbreak, and search term trend data. The best correlations between data sources were observed when the wastewater sewershed population had high overlap with the population included by other monitoring methods. The increased specificity and earlier detection of HuNoV GII using wastewater compared to other data, and the ability to make this data available to healthcare, public health, and the public in a timely manner, suggests that wastewater measurements of HuNoV GII will enhance existing public health surveillance efforts of norovirus.

Citation: Ammerman ML, Mullapudi S, Gilbert J, Figueroa K, de Paula Nogueira Cruz F, Bakker KM, et al. (2024) Norovirus GII wastewater monitoring for epidemiological surveillance. PLOS Water 3(1): e0000198. https://doi.org/10.1371/journal.pwat.0000198 Editor: Ricardo Santos, Universidade Lisboa, Instituto superior Técnico, PORTUGAL Received: May 14, 2023; Accepted: November 23, 2023; Published: January 18, 2024 Copyright: © 2024 Ammerman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: Most data for this work has been made available in the paper and in the supplementary S1 Text. Analysis code and additional data used for this work can be found at https://github.com/epimath/norovirus_gii_wastewater_monitoring. Funding: This study was supported by funding from the University of Michigan through the Public Health Infection Prevention and Response Advisory Committee (PHIPRAC - KRW and MCE co-PIs), and from the Michigan Department of Health & Human Services through the Michigan Sequencing Academic Partnership for Public Health Innovation and Response (MI-SAPPHIRE) grant (S MA-2022 (ELCEDE-UM) 10/01/2021 – 07/31/2024, (BF mPI) and wastewater surveillance program (SEWER network grant, KRW and MCE co-PIs). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared no competing interests exist.

Introduction SARS-CoV-2 demonstrated the utility of wastewater monitoring of pathogens, and catalyzed the infrastructure development needed to monitor other pathogens of public health importance. One such pathogen is norovirus, which causes more than 90% of epidemic non-bacterial gastroenteritis outbreaks [1]. Norovirus is estimated to result in 900 deaths, 109,000 hospitalizations, 465,000 emergency department visits, and 2.3 million clinic visits [2], annually in the United States, causing substantial health and economic burdens [3]. Norovirus, a genera in the Caliciviridae family, is a nonenveloped virus with a positive-sense RNA genome. There are ten genogroups of Norovirus, GI–GX, and 48 genotypes [4]. The GII genogroup, specifically variants of the GII.4 genotype, are the most common cause of norovirus disease worldwide [3]. The most common route of transmission is person-to-person, followed by contaminated food or water, and contact with contaminated fomites [5]. An estimated 30% of all norovirus infections and over 40% of GII.4 infections are asymptomatic [6]. Reinfection is common due to relatively short-term immunity and the diverse genogroups [7,8]. Norovirus is a “winter” pathogen, in the northern hemisphere most outbreaks occur between November and April [9]. Human norovirus (HuNoV) surveillance practices vary greatly across the US, and there is no requirement for local, territorial, or state agencies to report individual norovirus cases to the national system. Public health officials often rely on syndromic data, such as school and emergency department gastrointestinal illness reports, as early indicators of norovirus outbreaks. This syndromic data is not specific to norovirus and is often only easily available to public health officials in a timely and local manner. Health departments are encouraged to report all waterborne, foodborne, and enteric disease outbreaks, which would include norovirus outbreaks, to the National Reporting System (NORS) and norovirus outbreaks to Calicinet [10]. The Centers for Disease Control and Prevention (CDC) and health departments from fourteen states, including Michigan, participate in the Norovirus Sentinel Testing and Tracking (NoroSTAT) network. This data is specific to norovirus and is made publicly available; however, the lag between norovirus testing and reporting can be as long as a few weeks–unacceptably long for a highly transmissible virus. Furthermore, data aggregated across participating states has minimal value for informing communities and much of norovirus transmission goes undetected by conventional surveillance systems due to asymptomatic cases and cases that do not require clinical care. Research on alternatives to traditional surveillance includes wastewater monitoring and digital epidemiology [11]. Wastewater monitoring for HuNoV has the potential to provide more local, early-warning information to inform public health decision-making—potentially prior to clinically detected outbreaks. Additionally, wastewater data can easily be made publicly available. Unlike other surveillance data, it is not as biased by human care-seeking behavior and clinical testing. However, rather than replace conventional epidemiological monitoring methods, wastewater data provides an alternative data source to triangulate with existing imperfect clinical data streams. HuNoV is detectable in wastewater treatment plant (WWTP) influent [12–14] and studies have reported positive correlations between wastewater levels and conventional surveillance data, including confirmed HuNoV cases [15–17], gastrointestinal (GI) illness cases [18,19], and confirmed hospital cases [19,20]. A study in Japan found stronger correlations between wastewater levels and regional rather than national cases [18]. A recent study in the US showed population weighted average HuNoV GII RNA levels across 145 WWTPs positively correlated to national NoroSTAT clinical data [21]. Wastewater data led conventional surveillance data in some reports and not in others [16,18,20]. These discrepancies could be due to a number of reasons, including that the regions covered by the sewershed did not always correspond to the regions covered by the conventional surveillance data, differences between the study populations, the different types of surveillance data used between the studies (e.g., hospital HuNoV cases, confirmed GI cases, syndromic data, etc.), and the low temporal resolution of wastewater and conventional surveillance data used in some of the studies. This suggests a need to examine corresponding wastewater and epidemiological surveillance data for multiple populations at high resolution and concurrently against multiple sources of conventional norovirus surveillance data. Another new approach to understanding trends for unreported illnesses is digital epidemiology—the use of search data, social media, mobile phone networks, and other digital data not generated for a public health purpose to understand epidemiological patterns [22]. Digital epidemiology has increasingly been used to understand seasonal and other temporal trends of disease across locations using search term data [23,24], suggesting it could also provide a useful data source for early warning of norovirus increases. Google searches have been correlated with norovirus case data [11]; however, this data can be biased based on terminology, behavior, media coverage and access and often lacks spatial resolution down at the community level. This type of digital epidemiological data has not previously been compared with wastewater data, opening up the potential to build a comprehensive early warning system that bridges across multiple clinical, digital, and wastewater data sources. In this study, we explore cross-correlations between HuNoV GII wastewater data and syndromic, outbreak, and Google search term data at multiple spatial scales and locations over a one year period, enabling us to explore the potential for triangulation across clinical, outbreak, digital, and wastewater data to understand human norovirus patterns. We measured HuNoV GII levels at high temporal resolution in five WWTPs with different sizes and urban/rural composition, analyzing between two and seven samples per WWTP per week when HuNoV GII levels were elevated. We focused on HuNoV GII testing because 392 of the 440 US confirmed norovirus outbreaks recorded by Calicinet (or 89.1%) in 2021–2022 were HuNoV GII, and HuNoV GII has been shown to be more abundant than HuNoV GI in wastewater in North America [12]. Overall, this study assessed the ability of wastewater monitoring of HuNoV GII to provide added value to public health surveillance in combination with other traditional epidemiological surveillance methods.

Methods The Michigan SARS-CoV-2 Epidemiology–Wastewater Evaluation and Reporting (SEWER) Network includes local partnerships between wastewater utilities, health departments, tribal communities, universities, and laboratories that are collecting and analyzing wastewater for SARS-CoV-2 across the state of Michigan. All SEWER network laboratories use influent samples and approximately the same RNA purification methods. This infrastructure allowed us to monitor samples for additional RNA viruses. Sample collection procedure Samples were provided by five WWTPs in southeast Michigan (Ann Arbor, Flint, Jackson, Tecumseh, and Ypsilanti; Table 1) based on agreements established in June 2021. WWTP personnel collected daily influent samples between 7/18/2021 and 7/14/2022, except Tecumseh where sample collection began 1/12/2022. Samples were collected by 24-hour composite samplers kept at 4°C, and 50 ml aliquots were delivered biweekly by courier on ice to the University of Michigan. Samples were stored at 4°C and processed within 120 hours of collection, with limited degradation of target RNAs expected over this time [25–27]. Information about each WWTP was obtained from the state of Michigan SWEEP website: https://www.michigan.gov/coronavirus/stats/wastewater-surveillance/dashboard/sentinel-wastewater-epidemiology-evaluation-project-sweepwebsite. PPT PowerPoint slide

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TIFF original image Download: Table 1. Information about wastewater catchment areas. https://doi.org/10.1371/journal.pwat.0000198.t001 Sample processing We concentrated viruses in 40 ml samples 8–40 fold using PEG precipitation as previously described with minor modifications to scale down the starting material [28]. Briefly, 50 ml of influent sample was pasteurized for 30 minutes at 56°C. PEG 8000 (Fisher Scientific #BP2331) and 5 M NaCl solution (Sigma-Aldrich #S6546) were added to 40 ml of pasteurized influent samples to final concentrations of 8% (w/vol) and 0.2 M respectively. Samples were gently mixed and incubated at 4ºC for at least 8 hours. Samples were spun down at 4700 x g for 45 minutes at 4ºC. The supernatant was removed, leaving 1–5 mls of concentrated precipitate, with a bias towards lower volumes for samples with less solids to increase likelihood of detection in samples with low virus levels. RNA extraction was performed with 200 ul of sample concentrate using the QIAmp Viral RNA Mini Kit (Qiagen Sciences, MD) with an elution volume of 80 ul of water. A single extraction was performed for each sample collected. Bovine coronavirus (BCoV) (Bovilis Coronavirus Vaccine, Merck Animal Health, NJ) was added to samples prior to RNA extraction reactions as a recovery control. Negative extraction controls and positive BCoV and HuNoV GII extraction controls were prepared. Non-infectious intact HuNoV GI and GII particles (Cat# NATNOV-6MC, Zeptometrix, Buffalo, NY) were used to confirm the efficacy of norovirus RNA extraction and the specificity of the HuNoV GII primer/probe set in ddPCR reactions. PEG precipitation and RNA extraction using the QIAmp Viral RNA kit have previously been shown to be effective methods for norovirus RNA purification [29]. Pepper mild mottle virus (PMMoV) was quantified as a measure of fecal content [30]. RT-ddPCR Assay details for RT-ddPCR are provided as per MIQE guidelines [31]. Reverse transcription—digital droplet PCR (RT-ddPCR) analysis of HuNoV GII was performed on new RNA samples using multiplexing (with SARS N1 and N2) or triplexing (with BCoV and PMMoV) from March 25, 2022 to July 14, 2022. RNA from samples July 18, 2021 to March 25, 2022 were freeze-thawed once prior to ddPCR, and had been stored at -80°C for 1–8 months. A total of 643 samples were tested, only 3 samples had no detectable HuNoV GII. Gene copies were quantified through one-step RT-ddPCR (n = 3) using the One-step RT-ddPCR Advanced kit for Probes (catalog #1864021, Bio-Rad, CA) with the method described by Flood et al with some modifications [28]. RT-ddPCR reactions were run at 50C for 60 minutes, 95C for 10 minutes, 40 cycles of 95C for 30 seconds and 56C for 1 minute, 98C for 10 minutes, and held at 4C. HuNoV GII-specific primers and probes (synthesized by Integrated DNA Technologies, IA) that target a 97 bp region of the ORF1-ORF2 region and were previously tested for ddPCR were used [32,33]. When HuNoV GII levels were low, 5 μl undiluted RNA was analyzed by multiplexing with the SARS-CoV-2 nucleocapsid 1 (N1) and nucleocapsid 2 (N2) gene targets designed by the US Centers for Disease Control and Prevention (CDC) [34] using the 5’-FAM tagged HuNoV GII probe at half the concentration of the N1 and N2 probes (125 nM for the HuNoV GII probe, 250 nM for SARS-CoV-2 probes). When HuNoV GII levels were high, samples were diluted 1:100 and 5 μl of the diluted RNA was analyzed by triplexing with BCoV and PMMoV targets using both 5’-FAM and 5’-HEX HuNoV GII probe at half the concentration of the BCoV and PMMoV probes (125 nM for both HuNoV GII probes, 250 nM for BCoV and PMMoV probes). Primer concentrations were all 900 nM. All sample and control reactions were run in triplicate. Droplet analysis was performed on the Bio-Rad QX200 droplet digital PCR systems (Bio-Rad, CA) with explanations of thresholding provided in S1 Text. The results from replicate wells were merged. Controls were run along with all PCR reactions and included non-template controls, extraction controls, and positive controls. The N1/N2 primer/probe stocks and positive PCR controls were provided by MSU Rose lab as part of the SEWER project. PMMoV detection methods had previously been established in the laboratory [35] with a published gene block control [30]. A 390 bp G-block (IDT) DNA, specific to the ORF1-2 region of Genebank sequence MT474038.1, was used as a PCR control for HuNoV GII (Table A in S1 Text). To test both the dynamic range of our target and the presence of inhibition in our samples, a dilution series of HuNoV GII DNA from 2,500 to 25 gene copies was added to both nuclease free water and wastewater sample RNA extract prior to performing ddPCR. The same sample RNA extract was tested without the HuNoV GII DNA spiked in to determine the background HuNoV GII RNA levels in the sample. We saw a clear relationship between the amount of HuNoV GII DNA spiked in the extract and the quantities detected in the blank and sample extracts. This indicated the absence of inhibition in the wastewater RNA extracts. Plates containing negative and no template control wells that had greater than 3 droplets were rerun or re-extracted, consistent with the protocol used by the SEWER network for SARS-CoV-2 ddPCR. Samples with less than 30% of the control reaction BCoV values were flagged for further analysis to determine if RNA degradation had occurred and re-extracted from PEG concentrates when appropriate. All primers, probes, and the HuNoV GII DNA control used are listed in Table A in S1 Text [30,32,34,36,37]. The limit of detection was established by serial dilution of the HuNoV GII DNA control and taking into account the limit of the blank (up to 3 positive droplets). Due to differences in sample concentration volumes after PEG precipitation (from 1–5 mls) the limit of detection varied from 6 x 103 to 2.4 x 104 gc/L. Syndromic, outbreak, and internet search term data School-reported GI illnesses and emergency department GI-related visit data were provided by local public health departments and are covered through a data use agreement between the Michigan Department of Health and Human Services (MDHHS) and the Wigginton and Eisenberg labs at the University of Michigan (DUA#: 23-UFA01896 with MDHHS). This project was determined to not be regulated by the UM institutional review board (IRB# HUM00218874). These syndromic data included only reports of symptoms associated with norovirus and no tested or confirmed norovirus cases. The collection and analysis methods used in this study complied with the terms and conditions for the source of the data. Per the CDC National Center for Health Statistics standard, to prevent disclosure of individual information, only data with > 10 cases was included. Weekly total norovirus outbreak values from September 25, 2021 through June 12, 2022 were obtained from the CDC NoroSTAT website (www.cdc.gov/norovirus/reporting/norostat) which compiles and reports outbreak data from 14 states including Michigan, representing about 25% of the US population. Google Trends records how often a term is searched for in a given region relative to its total search volume with the normalized value in a range from 0 to 100. We obtained time-series data for the search terms “norovirus”, “gastroenteritis”, “stomach flu” for the Metropolitan Detroit region and the state of Michigan from 1/1/2021-11/29/2022. Data were joined by wastewater sample collection date and search term data week. Search term values were rolled forward to apply to all dates in a given week for matching purposes. Statistical analyses We conducted statistical analyses with Graphpad Prism 9.4.1 except where otherwise noted. We determined the median and interquartile range of log-transformed weekly average HuNoV GII wastewater concentrations (gc/L) and the log-transformed weekly average HuNoV GII/PMMoV wastewater concentration ratios from each WWTP, from all WWTPs combined, and by season. To ensure a consistent set of methods and to make full use of the numerical values of the data, we used a Pearson correlation for all analyses, although we also evaluated a subset of variables with both Pearson and Spearman correlations and found similar results (files included in our public repository). We calculated Pearson correlations and cross correlations between wastewater HuNoV GII values and the number of school-reported GI illnesses, emergency department GI visits, and weekly total outbreaks from NoroSTAT. Negative cross correlation lag times indicate clinical data leads wastewater data and positive values indicate wastewater values lead (See S1 Text for details). Pearson correlations and cross correlations were also computed between HuNoV GII against Detroit and Michigan-wide search term trends using R version 4.0.3 (2020-10-10)—"Bunny-Wunnies Freak Out"; Copyright (C) 2020 The R Foundation for Statistical Computing; Platform: x86_64-w64-mingw32/x64 (64-bit).

Conclusions In this study we assess the comparability of high spatiotemporal resolution HuNoV GII levels in wastewater from 5 WWTPs, to syndromic, outbreak and search trend data over the span of a year. Our results suggest that wastewater monitoring of HuNoV GII leads or concurs with other epidemiological monitoring methods, but correlations between wastewater and other data sources varied by the degree of overlap between the sewershed and the population catchment of the other data source. For example, the cross correlation values obtained when comparing state syndromic data to wastewater HuNoV GII values varied greatly between WWTPs (0.48 to 0.88). The lowest value was seen for the JS WWTP, which collects from a population of only about 90,000 individuals and represents less than 1% of the state population. JS HuNoV GII wastewater values exhibited a sharp peak in early March, unlike many other WWTPs that had elevated levels over a more extended time period. This combination of low population overlap and pronounced outbreak peak in JS likely accounts for the lower correlation values seen for JS. Similar to the state-level syndromic data, NoroSTAT aggregates clinical data across large geographical areas. There is a broad interest in defining the lead time of wastewater data compared to conventional surveillance data. Due to their poor geographic overlap with community wastewater data, the aggregate state syndromic data and national NoroSTAT data are not ideal for assessing the potential lead times of wastewater data. Although the more conventional epidemiological approaches are valuable and can help with forecasting, wastewater-based surveillance can provide a more focused regional picture of the norovirus cases compared to the state data and NoroSTAT that covers such large areas. Overall, our results suggest that smaller populations and/or closer overlap between the wastewater and syndromic or case populations results in closer temporal correlation. However, there is a limit to how small a sewershed population can be before other factors such as large variability in signals and individual variations in shedding become an issue [40], and this may vary by pathogen. A limitation of this and future studies is the lack of gold standard case data for norovirus which required us to consider correlations across multiple epidemiological datasets to validate wastewater detection. Additional limitations include that several of our epidemiological data sets could not be resolved at the same spatial geography as our wastewater sewershed data (for example, the Google Trends data could only be resolved to the Detroit region rather than the catchment areas), potentially reducing correlations due to differences in the populations measured, and biasing our lead/lag times if norovirus spatial spread patterns led to one population experiencing increases in transmission patterns before the other population did. It should also be noted that we focused on HuNoV GII in wastewater and HuNoV GI is likely contributing to clinical values in many locations. Our study adds to the existing literature by considering: multiple treatment plants based in the US spanning a range of population density and urbanicity, high resolution time series data spanning over a year from 600+ wastewater samples, and comparing wastewater data to multiple epidemiological data sets—including taking a digital epidemiology approach [41]. The advantages of wastewater data compared to syndromic and case data include more timely availability and accessibility of data and ability to detect asymptomatic and mild cases who may not seek care. However, these benefits depend on testing frequency, timely reporting using public dashboards, and diversity and representation of the WWTPs being tested. Given that wastewater data for HuNoV GII correlated closely with multiple other syndromic, outbreak, and search term measures of norovirus activity, and did so either concurrently or as a leading indicator, when appropriately implemented wastewater surveillance of HuNoV GII can provide a useful early warning system and an complementary data stream with which to triangulate norovirus patterns—one that does not require healthcare seeking, clinical testing, or inference based on symptom patterns.

Supporting information S1 Text. Document contains supplementary figures and methods. Fig A. Comparison of HuNoV GII levels from 5 WWTPs in Michigan from 2021–2022. HuNoV was quantified in influent samples using ddPCR at least weekly and gene copies per liter (gc/L) were plotted over one year. The only exception is TM, where sample collection began later, in January 2022. B and C. Box-and-whisker plots of log10 (B) average weekly gc/L HuNoV values (median, IQR) or (C) HuNoV/PMMoV values for each WWTP for the entire test period. Fig B. Individual graphs of HuNoV GII levels from 5 WWTPs in Michigan from 2021–2022 analyzed by two different methods. HuNoV was quantified in influent samples using ddPCR at least weekly and gene copies per liter (gc/L), shown in orange, as well as HuNoV values normalized to the fecal indicator PMMoV, shown in blue, were plotted over time. The only exception is TM, where sample collection began later, in January 2022. Values from WWTPs in A. Ann Arbor (AA), B. Flint (FL), C. Jackson (JS), D. Tecumseh (TM), and E. area around Ypsilanti Community (YC). Fig C. Seasonal variations in PMMoV-normalized HuNoV GII levels in wastewater. A. HuNoV was quantified in influent samples using ddPCR at least weekly. The only exception is TM, where sample collection began later, in January 2022. The weekly averages of HuNoV/PMMoV were analyzed and a box and whisker plot were used to display the log 10 values (median, IQR). B. Seasonal HuNoV GII values (median, IQR) for each individual WWTP are presented using box-and-whisker plots. Note, no fall values for TM were obtained. The definition of the seasons is meteorological, beginning on the 1st day of the equinoxes or solstices. Fig D. Seasonal variations in HuNoV GII levels (gc/L) in wastewater. A. HuNoV was quantified in influent samples using ddPCR at least weekly. The only exception is TM, where sample collection began later, in January 2022. The weekly averages of HuNoV GII GC/L were analyzed and a box and whisker plot were used to display the log 10 values (median, ICR). B. Seasonal HuNoV GII values (median, IQR) for all WWTPs are presented using box-and-whisker plots. Note, no fall values for TM were obtained. Fig E. Cross correlations of HuNoV GII wastewater values in gc/L with weekly school reported gastrointestinal illnesses. Graphs show Pearson’s correlation coefficients (r) and probability values (p) determined by comparing weekly average wastewater data in HuNoV II gc/L, to school reported GI illnesses. A. Weekly number of school reported GI illnesses in TM schools was compared to weekly average TM WWTP HuNoV GII values. B. Weekly County-level school reported GI illness values normalized for attendance were compared to weekly average WWTP HuNoV GII values for AA and YC. Cross correlations were tested for a lead (14 or 7 days before), same timing, or a lag (7, 14, 21 and in some cases 28 and 35 days later) in school reported GI illnesses compared to HuNoV GII values. Note: * = p<0.05, ** = p<0.01, and *** = p < .001. Fig F. Correlation of HuNoV GII gc/L wastewater values with weekly GI related emergency department visits at regional and state levels. A. Graph showing Pearson’s correlation coefficients (r) and probability values (p) determined by comparing HuNoV GII wastewater values (in gc/L) from the TM, AA, and YC WWTPs to the weekly total GI related emergency department visits reported for patients from the corresponding counties (Lenawee and Washtenaw), and the TM zip code, for January–April 2022. B. Graph showing Pearson’s correlation coefficients (r) and probability values (p) determined by comparing wastewater data as in A, to the weekly percentage of GI related emergency department visits compared to total visits. Data was reported for patients from the TM WWTP zip code and associated county (Lenawee). State (MI) hospital values were also compared to HuNoV GII wastewater values from all 5 WWTPs for January–April 2022. Cross correlations were tested for a lead (14 or 7 days before), same timing, or a lag (7, 14, and 21 days). Note: * = p<0.05, ** = p<0.01, *** = p<0.001. Fig G. Area contained in google search trends dataset. A. Map showing the lower peninsula of Michigan. B. A zoomed in version of southeast Michigan with the region included in the Detroit Metro google search trend dataset in mauve. Each of our WWTP catchment areas is shown in gray and labeled. Base map tile/data was provided by (c) OpenStreetMap and contributors, CC-BY-SA [openstreetmap.org/copyright]. Fig H. Cross correlations comparing HuNoV GII/PMMoV values from 5 WWTPs and search term trends for “Norovirus” in the state of Michigan (left) and the Detroit Metro area (right). Pearson’s correlation coefficients (r) and probability values (p) were determined. Cross correlations were tested for a lead (-21, -14, -7 days), same timing, or a lag (7, 14, 21, days) in search term trends compared to HuNoV GII/PMMoV values. Note: * = p<0.05, ** = p<0.01, *** = p < .001. Fig I. Cross correlations comparing HuNoV GII/PMMoV values from 5 WWTPs and search term trends for “Stomach flu” in the state of Michigan (left) and the Detroit Metro area (right). Pearson’s correlation coefficients (r) and probability values (p) were determined. Cross correlations were tested for a lead (-21, -14, -7 days), same timing, or a lag (7, 14, 21, days) in search term trends compared to HuNoV GII/PMMoV values. Note: * = p<0.05, ** = p<0.01, *** = p < .001. Fig J. Correlations of WWTP data with “gastroenteritis” search term trends. Correlations were performed to determine the Pearson’s correlation coefficients (r) for HuNoV GII/PMMoV values for all 5 WWTPs (AA, FL, JS, TM, and YC) with search term trends for “Gastroenteritis” (GE) in the Detroit Metro (Det Met) area and Michigan (MI). Table A. Primers, probes, and positive control used in RT-ddPCR. https://doi.org/10.1371/journal.pwat.0000198.s001 (DOCX)

Acknowledgments We thank the Ann Arbor WWTP, Flint WWTP, Jackson WWTP, Tecumseh WWTP, and Ypsilanti Community WWTP, Lenawee County Health Department, Jackson County Health Department, Genesee County Health Department, and Washtenaw County Health Department, especially Kristen Schweighoefer, Laura Bauman, and Juan Marquez.

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