Heat inactivation of aqueous viable norovirus and MS2 bacteriophage.

AIMS: This study aimed to compare the heat inactivation kinetics of viable human norovirus with the surrogate, MS2 bacteriophage as well as assess the decay of the RNA signal. METHODS AND RESULTS: Human intestinal enteroids were used to analyze the heat inactivation kinetics of viable human norovirus compared to the surrogate MS2 bacteriophage, which was cultured using a plaque assay. Norovirus decay rates were 0.22 min-1, 0.68 min-1, and 1.11 min-1 for 50°C, 60°C, and 70°C, respectively, and MS2 bacteriophage decay rates were 0.0065 min-1, 0.045 min-1, and 0.16 min-1 for 50°C, 60°C, and 70°C, respectively. Norovirus had significantly higher decay rates than MS2 bacteriophage at all tested temperatures (P = .002-.007). No decrease of RNA titers as measured by reverse transcription-PCR for both human norovirus and MS2 bacteriophage over time was observed, indicating molecular methods do not accurately depict viable human norovirus after heat inactivation and treatment efficiency is underestimated. CONCLUSIONS: Overall, our data demonstrate that MS2 bacteriophage is a conservative surrogate to measure heat inactivation and potentially overestimates the infectious risk of norovirus. Furthermore, this study corroborates that measuring viral RNA titers, as evaluated by PCR methods, does not correlate with the persistence of viable norovirus under heat inactivation.

Wastewater surveillance for bacterial targets: current challenges and future goals.

Wastewater-based epidemiology (WBE) expanded rapidly in response to the COVID-19 pandemic. As the public health emergency has ended, researchers and practitioners are looking to shift the focus of existing wastewater surveillance programs to other targets, including bacteria. Bacterial targets may pose some unique challenges for WBE applications. To explore the current state of the field, the National Science Foundation-funded Research Coordination Network (RCN) on Wastewater Based Epidemiology for SARS-CoV-2 and Emerging Public Health Threats held a workshop in April 2023 to discuss the challenges and needs for wastewater bacterial surveillance. The targets and methods used in existing programs were diverse, with twelve different targets and nine different methods listed. Discussions during the workshop highlighted the challenges in adapting existing programs and identified research gaps in four key areas: choosing new targets, relating bacterial wastewater data to human disease incidence and prevalence, developing methods, and normalizing results. To help with these challenges and research gaps, the authors identified steps the larger community can take to improve bacteria wastewater surveillance. This includes developing data reporting standards and method optimization and validation for bacterial programs. Additionally, more work is needed to understand shedding patterns for potential bacterial targets to better relate wastewater data to human infections. Wastewater surveillance for bacteria can help provide insight into the underlying prevalence in communities, but much work is needed to establish these methods.IMPORTANCEWastewater surveillance was a useful tool to elucidate the burden and spread of SARS-CoV-2 during the pandemic. Public health officials and researchers are interested in expanding these surveillance programs to include bacterial targets, but many questions remain. The NSF-funded Research Coordination Network for Wastewater Surveillance of SARS-CoV-2 and Emerging Public Health Threats held a workshop to identify barriers and research gaps to implementing bacterial wastewater surveillance programs.

Environmental Persistence and Disinfection of Lassa Virus.

Lassa fever, caused by Lassa virus (LASV), is endemic to West Africa, where ≈300,000 illnesses and ≈5,000 deaths occur annually. LASV is primarily spread by infected multimammate rats via urine and fomites, highlighting the need to understand the environmental fate of LASV. We evaluated persistence of LASV Josiah and Sauerwald strains on surfaces, in aqueous solutions, and with sodium hypochlorite disinfection. Tested strains were more stable in deionized water (first-order rate constant [k] for Josiah, 0.23 days; for Sauerwald, k = 0.34 days) than primary influent wastewater (Josiah, k = 1.3 days; Sauerwald, k = 1.9 days). Both strains had similar decay rates on high-density polyethylene (Josiah, k = 4.3 days; Sauerwald, k = 2.3 days) and stainless steel (Josiah, k = 5.3 days; Sauerwald, k = 2.7 days). Sodium hypochlorite was highly effective at inactivating both strains. Our findings can inform future risk assessment and management efforts for Lassa fever.

Making waves: Establishing a modeling framework to evaluate novel targets for wastewater-based surveillance.

Wastewater-based surveillance (WBS) monitoring of pathogens circulating within a community provides an improved understanding of the occurrence and spread of infectious diseases. However, the potential suitability of WBS for novel disease targets is unclear, including many emerging and neglected diseases. The current ad hoc approach of conducting wastewater detection experiments on novel disease targets to determine their suitability for WBS monitoring is resource intensive and may stall investment in this promising technology. In addition, detections, or non-detections, without the context of disease prevalence and shedding by infected individuals are difficult to interpret upon initial WBS target development. In this paper, we present a WBS feasibility analysis framework to identify which diseases are theoretically appropriate for WBS applications and to improve the initial interpretation of target detections. We then discuss five primary factors that influence the probability of detection in WBS monitoring - genome shedding rate, infection rate, per capita wastewater usage, process limit of detection (PLOD), and the number of PCR replicates. Clarifying the relationships between these factors and the likelihood of detection enhances quantitative insights into applying WBS, guiding researchers and stakeholders into mitigating inherent uncertainties of wastewater monitoring and subsequent improvements in WBS outcomes, thereby supporting future investment and expansion of WBS research, especially in novel disease targets.

Stability of Monkeypox Virus in Body Fluids and Wastewater.

An outbreak of human mpox infection in nonendemic countries appears to have been driven largely by transmission through body fluids or skin-to-skin contact during sexual activity. We evaluated the stability of monkeypox virus (MPXV) in different environments and specific body fluids and tested the effectiveness of decontamination methodologies. MPXV decayed faster at higher temperatures, and rates varied considerably depending on the medium in which virus was suspended, both in solution and on surfaces. More proteinaceous fluids supported greater persistence. Chlorination was an effective decontamination technique, but only at higher concentrations. Wastewater was more difficult to decontaminate than plain deionized water; testing for infectious MPXV could be a helpful addition to PCR-based wastewater surveillance when high levels of viral DNA are detected. Our findings suggest that, because virus stability is sufficient to support environmental MPXV transmission in healthcare settings, exposure and dose-response will be limiting factors for those transmission routes.

Comparison of viral concentration techniques for native fecal indicators and pathogens from wastewater.

Viral pathogens are typically dilute in environmental waters, necessitating a concentration step prior to subsequent quantification or analysis. Historically, studies on viral concentration efficiency have been done by spiking known viruses into the sample; however, spike-in controls may not have the same behavior as "native" viruses exposed to environmental conditions. In this study, four concentration methods, including polyethylene glycol precipitation (PEG), skimmed milk flocculation (SMF), pH drop followed by filtration through a 0.45 µm filter (pH), and centrifugation using an Amicon filter (Amicon), were evaluated to concentrate native viral targets in wastewater. Viral targets included both indicators (crAssphage and pepper mild mottle virus) and pathogens (adenovirus, norovirus GII, human polyomavirus, and SARS-CoV-2) in addition to a bacterial marker (HF183). A non-native spike-in control was also added to compare native and spike-in recoveries. Recovery varied widely across targets and methods, ranging from 0.1 to 39.3 %. The Amicon method was the most broadly effective concentration for recovery efficiency. For the lowest-titer target, the PEG method resulted in the lowest number of non-detections, with 96.7 % positive detections for SARS-CoV-2, compared to 66.7 %, 80 %, and 76.7 % positive detections for SMF, pH, and Amicon, respectively. The non-native spike-ins chosen were only representative of a few native recovery trends, varying by both target and concentration method, and consistently under or over-estimated recovery. Overall, this study suggests the utility of including native targets in viral concentration evaluation and determining the efficiency of concentration methods for a specific target of interest.

Impact of fixture purging on water age and excess water usage, considering stochastic water demands.

Higher water ages are linked with water quality decline as chlorine dissipates, temperatures become more favorable for microbial growth, and metals and organic matter leach from the pipes. Water fixtures with automated purging devices can limit water age in premise plumbing systems, but also increase water use. To develop purging strategies that lower age while also minimizing water use, the stochastic nature of water demands must be considered. In this research, a hydraulic plumbing network model, with stochastic demands at fixtures, was used to compare water age and water use for five purging conditions: purging at regular intervals, "smart" purging (considering the time of last use), purging with different volumes of water, purging at different fixtures, and the purging with different levels of home occupancy. Higher purging frequency and volume resulted in lower water ages, but higher water use. Purging greatly reduced the variability in water ages, avoiding extreme ages entirely. Water age was minimized by scheduling the purging around occupancy behavior, such as before the occupants wake up or return from work. Scheduled purging used more water than smart purging. Purging after 12 h of nonuse used only 55% of the additional water required for purging every 12 h. Purging after 24 h of nonuse at the kitchen tap and shower used only 38% of the additional water required for purging every 24 h, while maintaining lower water ages and removing the variability in water ages. While larger purging volumes had a greater impact on water age, there were diminishing returns. Purging has a larger impact on low-occupancy homes because fixtures have less frequent use. Overall, this research provides a methodology to compare purging strategies that minimize both water age and water use. While the numerical results presented here are only valid for the specific layout and usage habits, they provide insights and trends applicable to other cases.

Environmental DNA (eDNA) removal rates in streams differ by particle size under varying substrate and light conditions.

The use of environmental DNA (eDNA) as a sampling tool offers insights into the detection of invasive and/or rare aquatic species and enables biodiversity assessment without traditional sampling approaches, which are often labor-intensive. However, our understanding of the environmental factors that impact eDNA removal (i.e., how rapidly eDNA is removed from the water column by the combination of decay and physical removal) in flowing waters is limited. This limitation constrains predictions about the location and density of target organisms after positive detection. To address this question, we spiked Common Carp (Cyprinus carpio) eDNA into recirculating mesocosms (n = 24) under varying light (shaded versus open) and benthic substrate conditions (no substrate, bare substrate, and biofilm-colonized substrate). We then collected water samples from each mesocosm at four time points (40 min, 6 h, 18 h, and 48 h), and sequentially filtered the samples through 10, 1.0, and 0.2 µm filters to quantify removal rates for different eDNA particle sizes under varying light and substrate conditions. Combining all size classes, total eDNA removal rates were higher for mesocosms with biofilm-colonized substrate compared to those with no substrate or bare (i.e., no biofilm) substrate, which is consistent with previous findings linking biofilm colonization with increased eDNA removal and degradation. Additionally, when biofilm was present, light availability increased eDNA removal; eDNA levels fell below detection after 6-18 h for open mesocosms versus 18-48 h for shaded mesocosms. Among size classes, larger particles (>10 µm) were removed faster than small particles (1.0-0.2 µm). These results suggest that changes in the distribution of eDNA size classes over time (e.g., with downstream transport) and with differing environmental conditions could be used to predict the location of target organisms in flowing waters, which will advance the use of eDNA as a tool for species monitoring and management.

Suspended Materials Affect Particle Size Distribution and Removal of Environmental DNA in Flowing Waters.

Environmental DNA (eDNA) in aquatic systems is a complex mixture that includes dissolved DNA, intracellular DNA, and particle-adsorbed DNA. Information about the various components of eDNA and their relative proportions could be used to discern target organism abundance and location. However, a limited knowledge of eDNA adsorption dynamics and interactions with other materials hinders these applications. To address this gap, we used recirculating stream mesocosms to investigate the impact of suspended materials (fine particulate organic matter, plankton, clay, and titanium dioxide) on the eDNA concentration and particle size distribution (PSD) from two fish species in flowing water. Our findings revealed that eDNA rapidly adsorbs to other materials in the water column, affecting its concentration and PSD. Nonetheless, only particulate organic matter affected eDNA removal rate after 30 h. Moreover, we observed that the removal of larger eDNA components (≥10 µm) was more strongly influenced by physical processes, whereas the removal of smaller eDNA components was driven by biological degradation. This disparity in removal mechanisms between larger and smaller eDNA components could explain changes in eDNA composition over time and space, which have implications for modeling the spatial distribution and abundance of target species and optimizing eDNA detection in high turbidity systems.

Persistence of Antibiotic Resistance Genes Varies with Particle Size and Substrate Conditions in Recirculating Streams.

Antibiotic resistance (AR) determinants are enriched in animal manures, a significant portion of which is land-applied as a soil amendment or as fertilizer, leading to potential AR runoff and microbial pollution in adjacent surface waters. To effectively inform AR monitoring and mitigation efforts, a thorough understanding and description of the persistence and transport of manure-derived AR in flowing waters are needed. We used experimental recirculating mesocosms to assess water-column removal rates of antibiotic resistance genes (ARGs) originating from a cow manure slurry collected from a dairy farm. We quantified the effect of three benthic (i.e., bottom) substrate variations and particle sizes of manure slurry on water column removal rates. Overall, we observed variation in ARG behavior across substrate treatments and particle sizes. For ARGs associated with small particles, removal rates were higher in mesocosms with a substrate. tetW was typically removed at the highest rates across particle size and treatment, followed by ermB and blaTEM. Our data suggests that both substrate character and particle size exert control on the fate and transport of ARGs in surface waters, laying the foundation for future research in this area to establish a predictive framework for AR persistence and fate in flowing waters.

Show us the data: global COVID-19 wastewater monitoring efforts, equity, and gaps.

A year since the declaration of the global coronavirus disease 2019 (COVID-19) pandemic, there were over 110 million cases and 2.5 million deaths. Learning from methods to track community spread of other viruses such as poliovirus, environmental virologists and those in the wastewater-based epidemiology (WBE) field quickly adapted their existing methods to detect SARS-CoV-2 RNA in wastewater. Unlike COVID-19 case and mortality data, there was not a global dashboard to track wastewater monitoring of SARS-CoV-2 RNA worldwide. This study provides a 1-year review of the "COVIDPoops19" global dashboard of universities, sites, and countries monitoring SARS-CoV-2 RNA in wastewater. Methods to assemble the dashboard combined standard literature review, Google Form submissions, and daily, social media keyword searches. Over 200 universities, 1400 sites, and 55 countries with 59 dashboards monitored wastewater for SARS-CoV-2 RNA. However, monitoring was primarily in high-income countries (65%) with less access to this valuable tool in low- and middle-income countries (35%). Data were not widely shared publicly or accessible to researchers to further inform public health actions, perform meta-analysis, better coordinate, and determine equitable distribution of monitoring sites. For WBE to be used to its full potential during COVID-19 and beyond, show us the data.

Simple fabrication of an electrospun polystyrene microfiber filter that meets N95 filtering facepiece respirator filtration and breathability standards.

During the global spread of COVID-19, high demand and limited availability of melt-blown filtration material led to a manufacturing backlog of N95 Filtering Facepiece Respirators (FFRs). This shortfall prompted the search for alternative filter materials that could be quickly mass produced while meeting N95 FFR filtration and breathability performance standards. Here, an unsupported, nonwoven layer of uncharged polystyrene (PS) microfibers was produced via electrospinning that achieves N95 performance standards based on physical parameters (e.g., filter thickness) alone. PS microfibers 3-6 µm in diameter and deposited in an ~5 mm thick filter layer are favorable for use in FFRs, achieving high filtration efficiencies (≥97.5%) and low pressure drops (≤15 mm H2O). The PS microfiber filter demonstrates durability upon disinfection with hydroxyl radicals (•OH), maintaining high filtration efficiencies and low pressure drops over six rounds of disinfection. Additionally, the PS microfibers exhibit antibacterial activity (1-log removal of E. coli) and can be modified readily through integration of silver nanoparticles (AgNPs) during electrospinning to enhance their activity (≥3-log removal at 25 wt% AgNP integration). Because of their tunable performance, potential reusability with disinfection, and antimicrobial properties, these electrospun PS microfibers may represent a suitable, alternative filter material for use in N95 FFRs.

Particle size influences decay rates of environmental DNA in aquatic systems.

Environmental DNA (eDNA) analysis is a powerful tool for remote detection of target organisms. However, obtaining quantitative and longitudinal information from eDNA data is challenging, requiring a deep understanding of eDNA ecology. Notably, if the various size components of eDNA decay at different rates, and we can separate them within a sample, their changing proportions could be used to obtain longitudinal dynamics information on targets. To test this possibility, we conducted an aquatic mesocosm experiment in which we separated fish-derived eDNA components using sequential filtration to evaluate the decay rate and changing proportion of various eDNA particle sizes over time. We then fit four alternative mathematical decay models to the data, building towards a predictive framework to interpret eDNA data from various particle sizes. We found that medium-sized particles (1-10 µm) decayed more slowly than other size classes (i.e., <1 and > 10 µm), and thus made up an increasing proportion of eDNA particles over time. We also observed distinct eDNA particle size distribution (PSD) between our Common carp and Rainbow trout samples, suggesting that target-specific assays are required to determine starting eDNA PSDs. Additionally, we found evidence that different sizes of eDNA particles do not decay independently, with particle size conversion replenishing smaller particles over time. Nonetheless, a parsimonious mathematical model where particle sizes decay independently best explained the data. Given these results, we suggest a framework to discern target distance and abundance with eDNA data by applying sequential filtration, which theoretically has both metabarcoding and single-target applications.

Quantitative risk assessment of COVID-19 aerosol transmission indoors: a mechanistic stochastic web application.

An increasing body of literature suggests that aerosol inhalation plays a primary role in COVID-19 transmission, particularly in indoor settings. Mechanistic stochastic models can help public health professionals, engineers, and space planners understand the risk of aerosol transmission of COVID-19 to mitigate it. We developed such model and a user-friendly web application to meet the need of accessible risk assessment tools during the COVID-19 pandemic. We built our model based on the Wells-Riley model of respiratory disease transmission, using quanta emission rates obtained from COVID-19 outbreak investigations. In this report, three modelled scenarios were evaluated and compared to epidemiological studies looking at similar settings: classrooms, weddings, and heavy exercise sessions. We found that the risk of long-range aerosol transmission increased 309-332% when people were not wearing masks, and 424-488% when the room was poorly ventilated in addition to no masks being worn across the scenarios. Also, the risk of transmission could be reduced by ∼40-60% with ventilation rates of 5 ACH for 1-4 h exposure events, and ∼70% with ventilation rates of 10 ACH for 4 h exposure events. Relative humidity reduced the risk of infection (inducing viral inactivation) by a maximum of ∼40% in a 4 h exposure event at 70% RH compared to a dryer indoor environment with 25% RH. Our web application has been used by more than 1000 people in 52 countries as of September 1st, 2021. Future work is needed to obtain SARS-CoV-2 dose-response functions for more accurate risk estimates.

Monkeypox outbreak: Wastewater and environmental surveillance perspective.

Monkeypox disease (MPXD), a viral disease caused by the monkeypox virus (MPXV), is an emerging zoonotic disease endemic in some countries of Central and Western Africa but seldom reported outside the affected region. Since May 2022, MPXD has been reported at least in 74 countries globally, prompting the World Health Organization to declare the MPXD outbreak a Public Health Emergency of International Concern. As of July 24, 2022; 92 % (68/74) of the countries with reported MPXD cases had no historical MPXD case reports. From the One Health perspective, the spread of MPXV in the environment poses a risk not only to humans but also to small mammals and may, ultimately, spread to potent novel host populations. Wastewater-based surveillance (WBS) has been extensively utilized to monitor communicable diseases, particularly during the ongoing COVID-19 pandemic. It helped in monitoring infectious disease caseloads as well as specific viral variants circulating in communities. The detection of MPXV DNA in lesion materials (e.g. skin, vesicle fluid, crusts), skin rashes, and various body fluids, including respiratory and nasal secretions, saliva, urine, feces, and semen of infected individuals, supports the possibility of using WBS as an early proxy for the detection of MPXV infections. WBS of MPXV DNA can be used to monitor MPXV activity/trends in sewerage network areas even before detecting laboratory-confirmed clinical cases within a community. However, several factors affect the detection of MPXV in wastewater including, but not limited to, routes and duration time of virus shedding by infected individuals, infection rates in the relevant affected population, environmental persistence, the processes and analytical sensitivity of the used methods. Further research is needed to identify the key factors that impact the detection of MPXV biomarkers in wastewater and improve the utility of WBS of MPXV as an early warning and monitoring tool for safeguarding human health. In this review, we shortly summarize aspects of the MPXV outbreak relevant to wastewater monitoring and discuss the challenges associated with WBS.

Looking Forward: The Role of Academic Researchers in Building Sustainable Wastewater Surveillance Programs.

BACKGROUND: In just over 2 years, tracking the COVID-19 pandemic through wastewater surveillance advanced from early reports of successful SARS-CoV-2 RNA detection in untreated wastewater to implementation of programs in at least 60 countries. Early wastewater monitoring efforts primarily originated in research laboratories and are now transitioning into more formal surveillance programs run in commercial and public health laboratories. A major challenge in this progression has been to simultaneously optimize methods and build scientific consensus while implementing surveillance programs, particularly during the rapidly changing landscape of the pandemic. Translating wastewater surveillance results for effective use by public health agencies also remains a key objective for the field. OBJECTIVES: We examined the evolution of wastewater surveillance to identify model collaborations and effective partnerships that have created rapid and sustained success. We propose needed areas of research and key roles academic researchers can play in the framework of wastewater surveillance to aid in the transition from early monitoring efforts to more formalized programs within the public health system. DISCUSSION: Although wastewater surveillance has rapidly developed as a useful public health tool for tracking COVID-19, there remain technical challenges and open scientific questions that academic researchers are equipped to address. This includes validating methodology and backfilling important knowledge gaps, such as fate and transport of surveillance targets and epidemiological links to wastewater concentrations. Our experience in initiating and implementing wastewater surveillance programs in the United States has allowed us to reflect on key barriers and draw useful lessons on how to promote synergy between different areas of expertise. As wastewater surveillance programs are formalized, the working relationships developed between academic researchers, commercial and public health laboratories, and data users should promote knowledge co-development. We believe active involvement of academic researchers will contribute to building robust surveillance programs that will ultimately provide new insights into population health. https://doi.org/10.1289/EHP11519.

Comparison of RT-dPCR and RT-qPCR and the effects of freeze-thaw cycle and glycine release buffer for wastewater SARS-CoV-2 analysis.

Public health efforts to control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic rely on accurate information on the spread of the disease in the community. Acute and surveillance testing has been primarily used to characterize the extent of the disease. However, obtaining a representative sample of the human population is challenging because of limited testing capacity and incomplete testing compliance. Wastewater-based epidemiology is an agnostic alternative to surveillance testing that provides an average sample from the population served by the treatment facility. We compare the performance of reverse transcription quantitative PCR (RT-qPCR) and reverse transcription digital droplet PCR (RT-dPCR) for analysis of SARS-CoV-2 RNA in a regional wastewater treatment facility in northern Indiana, USA from the earliest stages of the pandemic. 1-L grab samples of wastewater were clarified and concentrated. Nucleic acids were extracted from aliquots and analyzed in parallel using the two methods. Synthetic viral nucleic acids were used for method development and generation of add-in standard-curves. Both methods were highly sensitive in detecting SARS-CoV-2 in wastewater, with detection limits as low as 1 copy per 500 mL wastewater. RT-qPCR and RT-dPCR provided essentially identical coefficients of variation (s/[Formula: see text] = 0.15) for triplicate measurements made on wastewater samples taken on 16 days. We also observed a sevenfold decrease in viral load from a grab sample that was frozen at - 80 °C for 92 days compared to results obtained without freezing. Freezing samples before analysis should be discouraged. Finally, we found that treatment with a glycine release buffer resulted in a fourfold inhibition in RT-qPCR signal; treatment with a glycine release buffer also should be discouraged. Despite their prevalence and convenience in wastewater analysis, glycine release and freezing samples severely and additively (~ tenfold) degraded recovery and detection of SARS-CoV-2.

Comparison of RT-qPCR and RT-dPCR Platforms for the Trace Detection of SARS-CoV-2 RNA in Wastewater.

We compared reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RT digital PCR (RT-dPCR) platforms for the trace detection of SARS-CoV-2 RNA in low-prevalence COVID-19 locations in Queensland, Australia, using CDC N1 and CDC N2 assays. The assay limit of detection (ALOD), PCR inhibition rates, and performance characteristics of each assay, along with the positivity rates with the RT-qPCR and RT-dPCR platforms, were evaluated by seeding known concentrations of exogenous SARS-CoV-2 in wastewater. The ALODs using RT-dPCR were approximately 2-5 times lower than those using RT-qPCR. During sample processing, the endogenous (n = 96) and exogenous (n = 24) SARS-CoV-2 wastewater samples were separated, and RNA was extracted from both wastewater eluates and pellets (solids). The RT-dPCR platform demonstrated a detection rate significantly greater than that of RT-qPCR for the CDC N1 and CDC N2 assays in the eluate (N1, p = 0.0029; N2, p = 0.0003) and pellet (N1, p = 0.0015; N2, p = 0.0067) samples. The positivity results also indicated that for the analysis of SARS-CoV-2 RNA in wastewater, including the eluate and pellet samples may further increase the detection sensitivity using RT-dPCR.

Wastewater-based epidemiological surveillance to monitor the prevalence of SARS-CoV-2 in developing countries with onsite sanitation facilities.

Wastewater-based epidemiology (WBE) has emerged as a valuable approach for forecasting disease outbreaks in developed countries with a centralized sewage infrastructure. On the other hand, due to the absence of well-defined and systematic sewage networks, WBE is challenging to implement in developing countries like Bangladesh where most people live in rural areas. Identification of appropriate locations for rural Hotspot Based Sampling (HBS) and urban Drain Based Sampling (DBS) are critical to enable WBE based monitoring system. We investigated the best sampling locations from both urban and rural areas in Bangladesh after evaluating the sanitation infrastructure for forecasting COVID-19 prevalence. A total of 168 wastewater samples were collected from 14 districts of Bangladesh during each of the two peak pandemic seasons. RT-qPCR commercial kits were used to target ORF1ab and N genes. The presence of SARS-CoV-2 genetic materials was found in 98% (165/168) and 95% (160/168) wastewater samples in the first and second round sampling, respectively. Although wastewater effluents from both the marketplace and isolation center drains were found with the highest amount of genetic materials according to the mixed model, quantifiable SARS-CoV-2 RNAs were also identified in the other four sampling sites. Hence, wastewater samples of the marketplace in rural areas and isolation centers in urban areas can be considered the appropriate sampling sites to detect contagion hotspots. This is the first complete study to detect SARS-CoV-2 genetic components in wastewater samples collected from rural and urban areas for monitoring the COVID-19 pandemic. The results based on the study revealed a correlation between viral copy numbers in wastewater samples and SARS-CoV-2 positive cases reported by the Directorate General of Health Services (DGHS) as part of the national surveillance program for COVID-19 prevention. The findings of this study will help in setting strategies and guidelines for the selection of appropriate sampling sites, which will facilitate in development of comprehensive wastewater-based epidemiological systems for surveillance of rural and urban areas of low-income countries with inadequate sewage infrastructure.