Validation of Wastewater-Based Epidemiology Model Predictions and the Influence of Super-Shedders and Sewage Dynamics Using the Fecal Indicators Pepper Mild Mottle Virus and Carjivirus.

Wastewater-based epidemiology (WBE) emerged during the coronavirus disease 2019 (COVID-19) pandemic as a scalable and broadly applicable method for community-level monitoring of infectious disease burden. The lack of high-resolution fecal shedding data for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) limits our ability to link WBE measurements to disease burden. In this study, we present longitudinal, quantitative fecal shedding data for SARS-CoV-2 RNA, as well as for the commonly used fecal indicators pepper mild mottle virus (PMMoV) RNA and crAss-like phage (crAssphage) DNA. The shedding trajectories from 48 SARS-CoV-2-infected individuals suggest a highly individualized, dynamic course of SARS-CoV-2 RNA fecal shedding. Of the individuals that provided at least three stool samples spanning more than 14 days, 77% had one or more samples that tested positive for SARS-CoV-2 RNA. We detected PMMoV RNA in at least one sample from all individuals and in 96% (352/367) of samples overall. CrAssphage DNA was detected in at least one sample from 80% (38/48) of individuals and was detected in 48% (179/371) of all samples. The geometric mean concentrations of PMMoV and crAssphage in stool across all individuals were 8.7 × 104 and 1.4 × 104 gene copies/milligram-dry weight, respectively, and crAssphage shedding was more consistent for individuals than PMMoV shedding. These results provide us with a missing link needed to connect laboratory WBE results with mechanistic models, and this will aid in more accurate estimates of COVID-19 burden in sewersheds. Additionally, the PMMoV and crAssphage data are critical for evaluating their utility as fecal strength normalizing measures and for source-tracking applications. IMPORTANCE This research represents a critical step in the advancement of wastewater monitoring for public health. To date, mechanistic materials balance modeling of wastewater-based epidemiology has relied on SARS-CoV-2 fecal shedding estimates from small-scale clinical reports or meta-analyses of research using a wide range of analytical methodologies. Additionally, previous SARS-CoV-2 fecal shedding data have not contained sufficient methodological information for building accurate materials balance models. Like SARS-CoV-2, fecal shedding of PMMoV and crAssphage has been understudied to date. The data presented here provide externally valid and longitudinal fecal shedding data for SARS-CoV-2, PMMoV, and crAssphage which can be directly applied to WBE models and ultimately increase the utility of WBE.

Emergence of novel human pathogens pose significant challenges to human health as highlighted by the SARS-CoV-2 pandemic. Wastewater based epidemiology (WBE) has previously been employed to identify viral pathogens and outbreaks by testing samples from regional wastewater treatment plants. Near source tracking (NST) allows for more targeted WBE by analysing samples from individual buildings such as schools or even individual floors such as in multi-floor office buildings. Despite the public health advantages of WBE, few strategies exist for optimising NST sampling methodologies. Therefore, we developed a protocol to evaluate virus detection in NST sampling using Pepper Mild Mottle Virus (PMMoV) as a proxy for RNA viruses. PMMoV is the most abundant enteric human associated RNA virus and is present in peppers/pepper-containing foods. Two bespoke TaqMan RT-PCR assays were developed to detect a PMMoV genomic 5' region and a capsid associated gene. To evaluate the protocol against field samples, pepper homogenates were flushed down an in-use toilet (Liverpool School of Tropical Medicine, UK) to spike wastewater with PMMoV on multiple days, and samples collected from two sewage access points to validate NST samplers. These wastewater samples were assessed for PMMoV based on Ct values and results compared to pepper and Tabasco derived PMMoV positive controls. Positive detection of PMMoV was comparable and consistent in ten independent samples across two NST samplers regardless of pepper homogenate spiking. We have developed two novel one step TaqMan assays that amplify both PMMoV targets in viral RNA extractions from peppers, Tabasco, and wastewater samples with cDNA synthesis through to RT-PCR results taking approximately 30 minutes. Pepper homogenate flushing was not required to detect PMMoV in our wastewater samples, however this strategy of flushing PMMoV containing materials outlined here could be valuable in assessing and validating NST in buildings with no previous or current sewage flow.

As wastewater-based epidemiology (WBE) broadens its focus to include prevalent diseases with significant global health impact, existing surveillance systems concentrate on sewer-based infrastructure, which excludes the 2.7 billion people using non-sewered systems. To address this gap, our study explores the potential of fecal sludge treatment plants (FSTPs) for WBE, emphasizing the stability of virus RNA targets within pooled fecal sludge. We screened fecal sludge from a centralized treatment facility in Dakar, Senegal for SARS-CoV-2, human norovirus (HuNoV), and microbial source trackers (MSTs) pepper mild mottle virus (PMMoV) and tomato brown rugose fruit virus (ToBRFV). Decay kinetics of genomic RNA markers from these viruses were examined at 4 °C, 15 °C, and 30 °C over 70 days. Results indicate high persistence of viral targets in fecal sludge (T90 value of 3.3 months for exogenous SARS-CoV-2 N1 and N2, 6.2 months for ToBRFV), with all targets detected throughout the 70-day experiment under various temperatures with limited decay (<1 log10 reduction). This study addresses a crucial gap in understanding virus persistence in on-site sanitation systems by providing essential decay rate constants for effective target detection. Our results indicate that sampling at centralized facilities treating fecal sludge from on-site sanitation could facilitate localized pathogen surveillance in low-income settings.HighlightsInvestigation of the persistence of SARS-CoV-2, HuNoV, PMMoV, and ToBRFV genomic RNA in pooled fecal sludge derived from on-site sanitation systems.Novel microbial source tracker (MST), ToBRFV, exhibited comparable abundance to PMMoV, a well-established MST, in fecal sludge.No significant decay observed for HuNoV and PMMoV over 70 days at all temperature conditions (4, 15, and 30 °C).SARS-CoV-2 N1 and N2 showed T90values of 3.3 months at 30 °C.Fecal sludge treatment plants offer a centralized sampling location for wastewater-based epidemiology, providing a strategic approach for monitoring public health.Graphical Abstract

Since the start of the coronavirus-19 pandemic, the use of wastewater-based epidemiology (WBE) for disease surveillance has increased throughout the world. Because wastewater measurements are affected by external factors, processing WBE data typically includes a normalization step in order to adjust wastewater measurements (e.g., viral ribonucleic acid (RNA) concentrations) to account for variation due to dynamic population changes, sewer travel effects, or laboratory methods. Pepper mild mottle virus (PMMoV), a plant RNA virus abundant in human feces and wastewater, has been used as a fecal contamination indicator and has been used to normalize wastewater measurements extensively. However, there has been little work to characterize the spatiotemporal variability of PMMoV in wastewater, which may influence the effectiveness of PMMoV for adjusting or normalizing WBE measurements. Here, we investigate its variability across space and time using data collected over a two-year period from sewage treatment plants across the United States. We find that most variation in PMMoV measurements can be attributed to longitude and latitude followed by site-specific variables. Further research into cross-geographical and -temporal comparability of PMMoV-normalized pathogen concentrations would strengthen the utility of PMMoV in WBE.

Since the coronavirus disease 2019 (COVID-19) pandemic, wastewater-based epidemiology (WBE) has been widely applied in many countries and regions for monitoring COVID-19 transmission in the population through testing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater. However, the amount of virus shed by individuals over time based on the stage of infection and accurate number of infections in the community creates challenges in predicting COVID-19 prevalence in the population and interpreting WBE results. In this study, we measured SARS-CoV-2, pepper mild mottle virus (PMMoV), and human mitochondrial DNA (mtDNA) in longitudinal fecal samples collected from 42 COVID-19 patients for up to 42 days after diagnosis. SARS-CoV-2 RNA was detected in 73.1% (19/26) of inpatient study participants in at least one of the collected fecal specimens during the sampling period. Most participants shed the virus within 3 weeks after diagnosis, but five inpatient participants still shed the virus between 20 and 60 days after diagnosis. The median concentration of SARS-CoV-2 in positive fecal samples was 1.08 × 105 genome copies (GC)/gram dry fecal material. PMMoV and mtDNA were detected in 99.4% (154/155) and 100% (155/155) of all fecal samples, respectively. The median concentrations of PMMoV RNA and mtDNA in fecal samples were 1.73 × 107 and 2.49 × 108 GC/dry gram, respectively. These results provide important information about the dynamics of fecal shedding of SARS-CoV-2 and two human fecal indicators in COVID-19 patients. mtDNA showed higher positive rates, higher concentrations, and less variability between and within individuals than PMMoV, suggesting that mtDNA could be a better normalization factor for WBE results than PMMoV.

COVID-19 has spurred growth in the science surrounding wastewater-based epidemiology (WBE) pertaining to the detection of severe acute respiratory virus 2 (SARS-CoV-2) in waste streams as an early warning signal for public health. However, the highly variable wastewater environment has made it difficult to standardize an approach for sampling and analysis, especially in locations using combined sewer infrastructure. This study addresses knowledge gaps of WBE via three specific aims: (1) to compare diurnal fluctuations of SARS-CoV-2 and the human fecal indicator, pepper mild mottle virus (PMMoV) in wastewater treatment plant (WWTP) influent samples collected during dry versus wet weather conditions; (2) to assess accuracy of grab versus 24-hour composite samples collected under variable flow conditions; and (3) to examine changes in wastewater influent composition associated with rainfall derived inflow and infiltration (RDII) and impacts to SARS-CoV-2 and PMMoV abundance. Using droplet digital polymerase chain reaction (ddPCR), both SARS-CoV-2 and PMMoV were quantified hourly at two WWTPs in West Virginia during three wet and three dry weather events. A parallel configuration of two automated samplers was deployed at each WWTP to collect: (a) 24 grab samples, collected hourly, and (b) an equivalent 24-hour composite. Wastewater physiochemistry metadata (chemical oxygen demand, ammonia, conductivity, total suspended solids, turbidity, pH, temperature, and influent flow) was also collected. Results provided evidence of the influence of site-specific factors on viral abundance, including the potential role of septage haulers in skewed viral RNA abundance and RDII as a driver of overall viral concentrations. Significantly lower concentrations of SARS-CoV-2 were observed during wet weather days at both WWTPs (Mann Whitney U, p<0.001). On dry days, composite concentrations of SARS-CoV-2 ranged from 81% less than to 190% greater than the median hourly concentration, while results from wet weather days were more variable, ranging from 29% less than to 1,372% greater than the median hourly concentration, indicating potential flushing of viral RNA and dilution of grab samples to a point of near non-detection. Several physiochemical parameters showed significant positive correlations to viral RNA concentration regardless of dry or wet weather (Spearman’s rank, p<0.01), but consistency of observations was not preserved between both WWTPs, suggesting site-specific factors such as infiltration rate and community infection prevalence play a crucial role in determining viral presence and abundance in WBE samples. Collectively, this study identifies crucial determinants for WBE sample collection and data validation within combined sewer systems to enhance accuracy in reporting, inform local public health intervention, and mitigate the spread of infectious disease across communities.

Wastewater surveillance has emerged as a cost-effective and equitable approach for tracking the spread of SARS-CoV-2. In this study, we monitored the prevalence of SARS-CoV-2 on a university campus over three years (2021–2023) using wastewater-based epidemiology (WBE). Wastewater samples were collected from 11 manholes on campus, each draining wastewater from a corresponding dormitory building, and viral RNA concentrations were measured using reverse transcription quantitative PCR (RT-qPCR). Weekly clinical case data were also obtained from the university health center. A strong positive and significant correlation was observed between Grab and Composite sampling methods, supporting their robustness as equally effective approaches for sample collection. Specifically, a strong correlation was observed between Aggie Village 4 Grab and Aggie Village 4 Composite samples (R² = 0.84, p = 0.00) and between Barbee Grab and Barbee Composite samples (R² = 0.80, p = 0.00). Additionally, higher viral RNA copies of SARS-CoV-2 (N1 gene) were detected during the Spring semester compared to the Fall and Summer semesters. Notably, elevations in raw N1 concentrations were observed shortly after the return of college students to campus, suggesting that these increases were predomi-nantly associated with students returning at the beginning of the Fall and Spring semesters (January and August). To account for variations in fecal loading, SARS-CoV-2 RNA concentrations were normalized using Pepper Mild Mottle Virus (PMMoV), a widely used viral fecal biomarker. However, normalization using PMMoV did not improve correlations between SARS-CoV-2 RNA levels and clinical case data. Despite these findings, our study did not establish WBE as a consistently reliable complement to clinical testing in a university campus setting, contrary to many retrospective studies. One key limitation was that numerous off-campus students did not contribute to the campus wastewater system corresponding to the monitored dormitories. However, some off-campus students were still subjected to clinical testing at the university health center under mandated pro-tocols. Moreover, the university health center discontinued reporting cases per dormitory after 2021, making direct comparisons more challenging. Nevertheless, this study highlights the continued value of WBE as a surveillance tool for monitoring infectious diseases and provides critical insights into its application in campus environments.

Wastewater surveillance has emerged as a cost-effective and equitable approach for tracking the spread of SARS-CoV-2. In this study, we monitored the prevalence of SARS-CoV-2 on a university campus over three years (2021-2023) using wastewater-based epidemiology (WBE). Wastewater samples were collected from 11 manholes on campus, each draining wastewater from a corresponding dormitory building, and viral RNA concentrations were measured using reverse transcription-quantitative PCR (RT-qPCR). Weekly clinical case data were also obtained from the university health center. A strong positive and significant correlation was observed between Grab and Composite sampling methods, supporting their robustness as equally effective approaches for sample collection. Specifically, a strong correlation was observed between Aggie Village 4 Grab and Aggie Village 4 Composite samples (R2 = 0.84, p = 0.00) and between Barbee Grab and Barbee Composite samples (R2 = 0.80, p = 0.00). Additionally, higher viral RNA copies of SARS-CoV-2 (N1 gene) were detected during the Spring semester compared to the Fall and Summer semesters. Notably, elevations in raw N1 concentrations were observed shortly after the return of college students to campus, suggesting that these increases were predominantly associated with students returning at the beginning of the Fall and Spring semesters (January and August). To account for variations in fecal loading, SARS-CoV-2 RNA concentrations were normalized using Pepper Mild Mottle Virus (PMMoV), a widely used viral fecal biomarker. However, normalization using PMMoV did not improve correlations between SARS-CoV-2 RNA levels and clinical case data. Despite these findings, our study did not establish WBE as a consistently reliable complement to clinical testing in a university campus setting, contrary to many retrospective studies. One key limitation was that numerous off-campus students did not contribute to the campus wastewater system corresponding to the monitored dormitories. However, some off-campus students were still subjected to clinical testing at the university health center under mandated protocols. Moreover, the university health center discontinued reporting cases per dormitory after 2021, making direct comparisons more challenging. Nevertheless, this study highlights the continued value of WBE as a surveillance tool for monitoring infectious diseases and provides critical insights into its application in campus environments.

Wastewater-based epidemiology (WBE) has become a valuable tool for monitoring the prevalence of SARS-CoV-2 on university campuses. However, concerns about effectiveness of raw sewage as a COVID-19 early warning system still exist, and it's not clear how useful normalization by simultaneous comparison of Pepper Mild Mottle Virus (PMMoV) is in addressing variations resulting from fecal discharge dilution. This study aims to contribute insights into these aspects by conducting an academic-year field trial at the student residences on the University of Tennessee, Knoxville campus, raw sewage. This was done to investigate the correlations between SARS-CoV-2 RNA load, both with and without PMMoV normalization, and various parameters, including active COVID-19 cases, self-isolations, and their combination among all student residents. Significant positive correlations between SARS-CoV-2 RNA load a week prior, during the monitoring week, and the subsequent week with active cases. Despite these correlations, normalization by PMMoV does not enhance these associations. These findings suggest the potential utility of SARS-CoV-2 RNA load as an early warning indicator and provide valuable insights into the application and limitations of WBE for COVID-19 surveillance specifically within the context of raw sewage on university campuses.

Plasmid pBI143, abundant in the human gut, is a promising human-specific fecal marker. However, studies on its optimal concentration methods, seasonal variations, and potential as a normalization parameter for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), remain limited. Among the three concentration methods compared, polyethylene glycol (PEG) precipitation and centrifugation demonstrated comparable efficiencies (9.3±0.6 and 9.2±0.6 log10 copies/L, respectively; n=8 each), outperforming membrane filtration (8.0±0.6 log10 copies/L; n=8). PEG precipitation was further applied to quantify pBI143, together with other human-specific fecal markers (crAssphage and pepper mild mottle virus (PMMoV)), in 52 wastewater samples collected weekly over a one year from a wastewater treatment plant in Yamanashi Prefecture, Japan, by quantitative polymerase chain reaction. The higher pBI143 concentrations (9.6±0.5 log10 copies/L) compared to PMMoV (8.2±0.2 log10 copies/L) and crAssphage (8.0±0.2 log10 copies/L) highlighted its potential as a robust marker for human fecal contamination. Unlike PMMoV and crAssphage that remained stable across seasons, pBI143 showed seasonal fluctuations, especially during summer and autumn, suggesting its greater sensitivity to environmental conditions. The study evaluated the suitability of pBI143, crAssphage, and PMMoV for normalizing SARS-CoV-2 concentrations in wastewater; however, non-normalized SARS-CoV-2 concentrations showed the highest correlation with COVID-19 cases (ρ=0.74), whereas normalization reduced this correlation (PMMoV-normalized, ρ=0.72; crAssphage-normalized, ρ=0.70; and pBI143-normalized, ρ=0.50), likely due to differences in the persistence and structural properties of the markers, indicating that these markers are less effective for SARS-CoV-2 normalization. This study underscores the promising utility of pBI143 in wastewater surveillance but highlights the need for further research across diverse regions to validate its applicability.

Understanding the persistence of SARS-CoV-2 biomarkers in wastewater should guide wastewater-based epidemiology users in selecting best RNA biomarkers for reliable detection of the virus during current and future waves of the pandemic. In the present study, the persistence of endogenous SARS-CoV-2 were assessed during one month for six different RNA biomarkers and for the pepper mild mottle virus (PMMoV) at three different temperatures (4, 12 and 20 °C) in one wastewater sample. All SARS-CoV-2 RNA biomarkers were consistently detected during 6 days at 4° and differences in signal persistence among RNA biomarkers were mostly observed at 20 °C with N biomarkers being globally more persistent than RdRP, E and ORF1ab ones. SARS-CoV-2 signal persistence further decreased in a temperature dependent manner. At 12 and 20 °C, RNA biomarker losses of 1-log10 occurred on average after 6 and 4 days, and led to a complete signal loss after 13 and 6 days, respectively. Besides the effect of temperature, SARS-CoV-2 RNA signals were more persistent in the particulate phase compared to the aqueous one. Finally, PMMoV RNA signal was highly persistent in both phases and significantly differed from that of SARS-CoV-2 biomarkers. We further provide a detailed overview of the latest literature on SARS-CoV-2 and PMMoV decay rates in sewage matrices.

A direct capture method for purification and detection of viral nucleic acid enables epidemiological surveillance of SARS-CoV-2

Urban wastewater-based epidemiology for multi-viral pathogen surveillance in the Valencian region, Spain

Wastewater-based epidemiology (WBE) is a practical approach for detecting the presence of SARS-CoV-2 infections and assessing the epidemic trend of the coronavirus disease 2019 (COVID-19). The purpose of this study was to evaluate the minimum sampling frequency required to properly identify the COVID-19 trend during the downward epidemic period when using a highly sensitive RNA detection method. WBE was conducted using the Efficient and Practical virus Identification System with ENhanced Sensitivity for Solids (EPISENS-S), a highly sensitive SARS-CoV-2 RNA detection method, at nine neighboring wastewater treatment plants (WWTPs). These WWTPs were in the same prefecture in Japan, and they had different sewer types, sampling methods, and sampling frequencies. The overall detection rate of SARS-CoV-2 RNA was 97.8 % during the entire study period when the geometric means of new COVID-19 cases per 100,000 inhabitants were between 3.3 and 7.7 in each WWTP. The maximum SARS-CoV-2 RNA concentration in wastewater was 2.14 × 104 copies/L, which corresponded to pepper mild mottle virus (PMMoV)–normalized concentrations of 6.54 × 10−3. We evaluated the effect of sampling frequencies on the probability of a significant correlation with the number of newly reported COVID-19 cases by hypothetically reducing the sampling frequency in the same dataset. When the wastewater sampling frequency occurred 5, 3, 2, and 1 times per week, these results exhibited significant correlations of 100 % (5/5), 89 % (8/9), 85 % (23/27), and 48 % (13/27), respectively. To achieve significant correlation with a high probability of over 85 %, a minimum sampling frequency of twice per week is required, even if sampling methods and sewer types are different. WBE using the EPISENS-S method and a sampling frequency of more than twice a week can be used to properly monitor COVID-19 wave epidemic trends, even during downward periods.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is known to be present in sewage, and wastewater-based epidemiology has attracted much attention. However, the physical partitioning of SARS-CoV-2 in wastewater and the removal efficiency of treatment systems require further investigation. This study aimed to investigate the detectability and physical partitioning of SARS-CoV-2 in wastewater and assess its removal in a large-scale septic tank employing anaerobic, anoxic, and oxic processes in a sequential batch reactor, which was installed in a coronavirus disease 2019 (COVID-19) quarantine facility. The amount of SARS-CoV-2 RNA in wastewater was determined with polyethylene glycol (PEG) precipitation followed by quantitative polymerase chain reaction (qPCR), and the association of SARS-CoV-2 with wastewater solids was evaluated by the effect of filtration prior to PEG precipitation (pre-filtration). The amount of SARS-CoV-2 RNA detected from pre-filtered samples was substantially lower than that of samples without pre-filtration. These results suggest that most SARS-CoV-2 particles in wastewater are associated with the suspended solids excluded by pre-filtration. The removal efficiency of SARS-CoV-2 in the septic tank was evaluated based on the SARS-CoV-2 RNA concentrations in untreated and treated wastewater, which was determined by the detection method optimized in this study. Escherichia coli and pepper mild mottle virus (PMMoV) were also quantified to validate the wastewater treatment system's performance. The mean log10 reduction values of SARS-CoV-2, E. coli, and PMMoV were 2.47 (range, 2.25–2.68), 2.81 (range, 2.45–3.18), and 0.66 (range, 0.61–0.70), respectively, demonstrating that SARS-CoV-2 removal by the wastewater treatment system was comparable to or better than the removal of fecal indicators. These results suggest that SARS-CoV-2 can be readily removed by the septic tank. This is the first study to determine the removal efficiency of SARS-CoV-2 in a facility-level sequencing batch activated sludge system.