key: cord-0740610-g0t4mlcn
authors: Mazumder, Payal; Dash, Siddhant; Honda, Ryo; Sonne, Christian; Kumar, Manish
title: Sewage surveillance for SARS-CoV-2: molecular detection, quantification and normalization factors
date: 2022-04-10
journal: Current Opinion in Environmental Science & Health
DOI: 10.1016/j.coesh.2022.100363
sha: 5558452296731d99225fcc98bf034a6b92f1f54e
doc_id: 740610
cord_uid: g0t4mlcn

The presence of severe acute respiratory syndrome coronavirus–2 (SARS-CoV-2) in wastewater systems provide a primary indication of the coronavirus disease 2019 (COVID-19) spread throughout communities worldwide. Droplet Digital polymerase chain reaction (dd-PCR) or reverse transcription-polymerase chain reaction (RT-PCR) administration of SARS-CoV-2 in wastewaters provides a reliable and efficient technology for gathering secondary local-level public health data. Often the accuracy of prevalence estimation is hampered by many methodological issues connected with wastewater surveillance. Still, more studies are needed to use and create efficient approaches for deciphering the actual SARS-CoV-2 indication from noise in the specimens/samples. Nearly 39–65% of positive patients and asymptomatic carriers expel the virus through their feces however, only ∼6% of the infected hosts eject it through their urine. COVID-19 positive patients can shed the remnants of SARS-CoV-2 RNA virus within the concentrations ∼103 – 108 copies/L. However, it can decrease up to 102 copies/L in wastewaters due to dilution. Environmental virology and microbiology laboratories play a significant role in the identification and analysis of SARS-CoV-2 ribonucleic acid (RNA) in waste and ambient waters worldwide. Virus extraction or recovery from the wastewater (However, due to a lack of knowledge, established procedures, and integrated quality assurance/quality control (QA/QC) approaches, the novel coronavirus RNA investigation for estimating current illnesses and predicting future outbreaks is insufficient and/or conducted inadequately. The present manuscript is a technical review of the various methods and factors considered during identification of SARS-CoV-2 genetic material in wastewaters and/or sludge, including tips and tricks to be taken care of during sampling, virus concentration, normalization, PCR inhibition and trend line smoothening when compared with clinically active/positive cases.

As of November 2021, more than 262 million confirmed cases of COVID-19 have appeared worldwide, triggering the ongoing pandemic. Wastewater-based epidemiology (WBE) has proved to be a swift and supplementary tool for health authorities worldwide to monitor COVID-19 and also track the newly emerging variants of concern. The use of WBE has previously been effective in monitoring a range of harmful viruses including Polio, Noro, Dengue [1] and recently SARS-CoV throughout the world [2] [3] [4] . It targets the residues of the viruses' DNA/RNA that function as the pathogens' population quantitation. Unlike other respiratory viruses, the SARS-CoV-2 virus is located in most infested people's gastrointestinal tracts and feces [5] . Its injection into the wastewater streams was found in the initial pandemic phases, even prior to the first case in the communities were discovered [6] . WBE, therefore, hold the capability of acting as an early warning system for the development of COVID-19 at a societal stage by analyzing the signals of the viral load in wastewater samples pooled by population [7] .

It is worth noting that changes in wastewater SARS-CoV-2 RNA content is not necessarily proportionate to changes in confirmed cases or incidence [8] [9] [10] [11] . This data implies that wastewater is a complicated matrix because of the variability in numerous aspects including volume and duration of individual virus shedding, rates of RNA degradation, and carrier movement. Also, clinically confirmed cases do not cover the entire population of infections but only in the tested population. Confirmed cases are dependent on a scale of clinical testing (more testing finds more infections) and selection of examinee groups (generally, positive ratio decreases when examinee is selected randomly or an examinee group becomes larger). These fluctuations in confirmed cases also cause gap between confirmed cases and wastewater epidemic data, as well as catchment features and experimental errors. Therefore, more profound knowledge is needed about the SARS-CoV-2 virus variability in wastewaters and how that corresponds to the actual occurrence or dominance of COVID-19 in the conducive population to ensure relevance of the COVID-19 WBE for public health policymaking.

Inconsistencies in sample collection and processing in laboratories, degradation of nucleic acid owing to transit time and sewer environments and dilution of signal due to fluctuations in precipitation and daytime flow are all measures of target signal inconsistency in wastewaters [12] [13] [14] .

To assess the congruency of viral RNA content in wastewater with clinical cases, [15] built a model that included additional factors of high ambiguity and variability, such as rate of flow and per capita wastewater output, and viral rate of shedding. Several normalizing strategies are studied to adjust for the variation in fecal material induced by dilution, primarily using the two most familiar human fecal viral indicators, i.e., cross-assembly phage (crAssphage) and pepper mild mottle virus (PMMoV) [16] . There are several limiting conditions in the detection of the novel coronavirus in wastewater systems. Municipal wastewater is complicated and unpredictable concoction encompassing innumerable microorganisms and latent inhibitors along with several variants/strains of the SARS-CoV-2 virus enhancing the pre-requisites for precise procedures. In addition, SARS-CoV-2 genetic material appears in wastewater in miniscule requiring the design of trials with lower detection limits [17] . Thus, standardization of protocols and stringent methodologies will decrease the probability of errors (false positive, false negative) and thereby aid public health decision making in reducing COVID-19 epidemics.

Here we review the recent literature on concentration and identification methods of SARS-CoV-2 viral RNA from wastewater to assess local COVID-19 outbreaks including PCR technology, PCR inhibition and virus normalization factors for WBE of SARS-CoV-2. In addition, we discuss the future implications of SARS-CoV-2 monitoring in wastewater; and how the use of vaccines may help to end the pandemic.

To get detectable levels of viral nucleic acid, large volume sampling of more than 1 L is required for the concentration step [18] . The sample technique and time of sampling are critical elements for using WBE since they can affect data interpretation and possible cross-study comparisons [19] . Majority of research have concentrated on water samples, both small and large grab samples [20] [21] [22] as well as time or flow proportional composite samples [22] [23] [24] .

Other research, on the other hand, have used techniques such as Moore swabs, a gauze pad suspended in flowing WW and then processed. Grabs may be less expensive and easier to conduct than composite samples, but they may also have a higher level of unpredictability.

This variability is primarily determined by the volumes utilized, the distance of WWTP and sewer due to the viable virus decay over time, the time of day selected, given variations in both water consumption and source strength, which are connected to bathroom habits. When infection frequency is little and/or the sampled population size is small, such as in near-source J o u r n a l P r e -p r o o f sampling, when samples are gathered upstream in the sewage network close to the discharge source, quantitative estimates of the number of people affected are likely to be difficult to come by (e.g. outside a building). Because contributing events (e.g., toilet flushes) are more discrete and non-aggregated, grab sampling risks missing the event, and composite samples may be severely diluted by analytes lacking wastewater in the latter instance, the likelihood of collecting a representative sample is low.

Several studies have compared the concentration methods for enveloped viruses, notably the novel coronavirus monitoring, in wastewater [25] [26] [27] [28] [29] . The majority of such studies counted on exogenic viral controls put in wastewater samples to test the effectiveness of the process, and they were complemented by several cautions and restrictions [30] . Technologies that concentrate and quantify SARS-CoV-2 in wastewater have been well studied. These approaches' dependability, repeatability, and sensitivity must be confirmed for a more significant usage of the wastewater data. This "pooling approach" is essential in areas where clinical testing rates for COVID-19 are low, resources are few, or the number of cases is unknown [31] . MS2, a non-enveloped bacteriophage frequently employed as a process control of enteric virus detection, is one of several ways to detect and estimate SARS-CoV-2 concentrations in wastewater (used for process control) [32] . Two more enveloped coronaviruses (BCoV and OC43) are employed further to understand the efficacy in detecting SARS-CoV-2 in wastewater of which, the Concentrating Pipette (C.P.)-based concentration approach is more successful than the ViroCap-based (VC-based) when it comes to recovery efficiency and speed. The CP approach is much less time consuming than the VC-based technique making the efficacy of MS2 recovery was two times greater with the C.P. technique (53.6%) than with the VC-based method (24.7%) [8] . When it came to retrieving encapsulated coronaviruses from wastewater, V.C. was less effective (BCoV: 7.2%). Solids are eliminated prior to viral concentration, which might be an issue with this quick C.P. approach since enveloped viruses have been discovered as increasingly connected to the surface of the particles present in wastewater compared to viruses that lacks envelopes, in prior research [33] . Virus recovery efficiency post fractionation, polyethylene glycol (PEG) precipitation and viral RNA extraction in vesicular stomatitis virus (VSV) spiked wastewater samples was done by [34] (Equation 1).

J o u r n a l P r e -p r o o f [35] devised the 4S method to extract SARS-CoV-2 RNA from wastewater using a vacuum column to concentrate and purify RNA in less than 3 hours. This approach helps researchers at the University of California, Berkeley, to detect SARS-CoV-2 envelope (E) and nucleocapsid (N) gene RNA and PMMoV RNA using RT-qPCR probe-mediated detection. Other existing concentration methods use chemical precipitation [36] , size exclusion [37] , adsorption through membranes [38] , ultracentrifugation [39] , flocculation [26] , or a combination of such technologies [40, 41] with wastewater input amounts ranging from 15 to 250 mL. The approach described by [42] , in which the amount of RNA of SARS-CoV-2 conducted from wastewater of 45 L quantity via electropositive cartridges, is the only large-volume concentration technique published in previous articles; nonetheless, being very labor intensive. Table 1 shows the various approaches by researchers reported recently on the wastewater monitoring methods of SARS-CoV-2.

In wastewater matrix/systems, detection of SARS-CoV-2 RNA starts with sampling from inside a wastewater network structure such as a wastewater pumping stations, manholes or an influent from sewage treatment plants (STPs) or close by building outlet discharge. Then, the virus is concentrated, extricated and the RNA is run through dd-PCR (does not require a standard plot) and/or RT-qPCR (centered on a standard plot) for identification and quantification of SARS-CoV-2 genetic material. Poor viral retrieval and/or testing of minor effective sample volume (ESV), inefficiency of extracting RNA, intensification-inhibition in the PCR assay, and inadequate sensitivity assessment are all issues that affect sample processing and analysis [41, 43] . A number of strategies for removing or inactivating PCR inhibiting entities have been documented, although none of them are always efficient [44] .

Process controls, also known as external/internal controls, should be used to evaluate the efficiency of methods. According to the materials used and the points at which the controls are spiked into the sample, there are mainly three types of controls: (1) whole process controls [45, 46] , which are added before viruses are concentrated from the sample; (2) molecular process controls [47] [48] [49] , which are added before nucleic acid extractions; and (3) (RT-)qPCR controls [50] [51] [52] , which are added before the PCR processes. The impacts of inhibitors are reflected in the recovery yields of the process controls, which helps to better comprehend the study's conclusions. Many prior studies have attempted to establish a threshold value for projected recovery of process controls in order to confirm the accuracy of viral detection;

however, as shown below, there is presently no consensus on that number [52] . 

Normalization is recently considered effective since viral concentration in wastewater is affected by dilution with non-fecal wastewater and quantity of fecal discharge at the corresponding time of sampling. Normalization concepts using biomarkers, higher frequency of sampling, sample replicate processing, and smoothening/forecasting have been used to address some of the drivers of variability in SARS-CoV-2 identification. Normalization with population, the target signal, or an endogenic biomarker, enables to minimize inconsistency in data and scale estimates for cross-sample and cross-location comparisons. To compute the per capita load [57] or a chemical parameter, standardized concentrations of wastewaters to population and flow rate across WBE experiments (e.g., caffeine) is used [34] . COVID-19

WBE assays typically relies on the detection of a non-enveloped plant virus viz., Pepper mild mottle virus (PMMoV) RNA detection, however its quantities in sewage fluctuate depending on the season and local food [34] . A non-enveloped DNA virus viz., cross-assembly phage (crAssphage), that contaminates the Bacteroides (commensal bacterium) present in human gut, is another normalization biomarker that has been widely put to use [58] . However, crAssphage shedding per person varies highly, this normalization technique can be applied for population size >5000. Furthermore, the 16S rRNA gene of the human specific HF183 Bacteroides is commonly utilized to identify fecal pollution in natural waters, and subsequent research [34] have focused on HF183 rRNA to improve the sensitivity of the assay. Finally, since it aims at human cells excreted through faeces, the 18S rRNA, ribosomal subunit (18S) present in humans was recommended as the biomarker for normalization [35] . Similar to normalizing the target signal, smoothing processes can aid in determining sequential patterns in the incidence of COVID-19. Although 7 day moving averages is frequently utilized to examine real-time clinical data patterns [62] , low frequency of wastewater sample collection (e.g., once or twice a week) makes it difficult to use such approach. As a result, smoothing approaches such as locally weighted scatter plot smoothing, which may be employed to lesser sampling frequency data and minimalize the damage to temporal resolution, are required [63] .

Nevertheless, there is no benchmark value for the bandwidth variable (similar to choosing a gap of 7 days for moving averages). Additionally, in research including lowess, the bandwidth selection technique is rarely mentioned [31, 64] . to allow recurrent wastewater assessments at small case numbers [65] . Researchers have calculated this value observationally using fewer data points by reporting the number of instances they could identify or quantify [66] . developed connections linking SARS-CoV-2 genetic material load in wastewater and pandemic health indices using modelling approaches such as distributed/fixed lag modelling, linear regression, and artificial neural networks. Such models [68, 69] can be very helpful in conducting risk assessment for future outbreaks.

Both methodological studies for testing SARS-CoV-2 in wastewaters and the effectuation of 

This paper discusses how public health decisions are made easy with wastewater-based epidemiology of SARS-CoV-2 virus

Public Health Benefits and Ethical Aspects in the Collection and Open Sharing of Wastewater-Based Epidemic Data on COVID-19

This paper discusses WBE data generation and interpretation and is crucial for this review

Wastewater SARS-CoV-2 monitoring as a community-level COVID-19 trend tracker and variants in Ohio, United States, Science of The Total Environment

Correlation of SARS-CoV-2 RNA in wastewater with COVID-19 disease burden in sewersheds

SARS-CoV-2 Titers in Wastewater Are Higher than Expected from Clinically Confirmed Cases

SARS-CoV-2 from faeces to wastewater treatment: What do we know? A review

Wastewater-based epidemiology-surveillance and early detection of waterborne pathogens with a focus on SARS-CoV-2, Cryptosporidium and Giardia

Modeling wastewater temperature and attenuation of sewage-borne biomarkers globally

Evaluation of Sampling, Analysis, and Normalization Methods for SARS-CoV-2 Concentrations in Wastewater to Assess COVID-19 Burdens in Wisconsin Communities

First confirmed detection of SARS-CoV-2 in untreated wastewater in Australia: A proof of concept for the wastewater surveillance of COVID-19 in the community

Persistence of emerging viral fecal indicators in largescale freshwater mesocosms

First proof of the capability of wastewater surveillance for COVID-19 in India through detection of genetic material of SARS-CoV-2, Science of The Total Environment

Comparison of Concentration Methods for Quantitative Detection of Sewage-Associated Viral Markers in Environmental Waters

Monitoring COVID-19 through SARS-CoV-2 quantification in wastewater: progress, challenges and prospects

Measurement of SARS-CoV-2 RNA in wastewater tracks community infection dynamics

SARS-CoV-2 RNA in wastewater anticipated COVID-19 occurrence in a low prevalence area

First detection of SARS-CoV-2 RNA in wastewater in North America: A study in Louisiana, USA, Science of The Total Environment

Development of a reproducible method for monitoring SARS-CoV-2 in wastewater

Detection of SARS-CoV-2 in raw and treated wastewater in Germany -Suitability for COVID-19 surveillance and potential transmission risks

Concentration methods for the quantification of coronavirus and other potentially pandemic enveloped virus from wastewater, Current Opinion in Environmental Science & Health

This paper discusses SARS-CoV-2 virus concentration techniques from wastewater and is very helpful to understand the methods and it is one of the basis on which the present review is written

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

Comparison of approaches to quantify SARS-CoV-2 in wastewater using RT-qPCR: Results and implications from a collaborative inter-laboratory study in Canada

Approaches applied to detect SARS-CoV-2 in wastewater and perspectives post-COVID-19

Evaluation of viral concentration methods for SARS-CoV-2 recovery from wastewaters

Challenges in Measuring the Recovery of SARS-CoV-2 from Wastewater

This paper discusses the challenges in measuring SARS-CoV-2 concentration and process recovery

Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance

This paper has very good essence and discusses how errors can be minimized during WBE of SARS-CoV-2. Hence, it is very useful for the researchers to know the important points to keep in mind for precise and accurate results while conducting wastewater surveillance

Aerosolization of Ebola Virus Surrogates in Wastewater Systems

Survivability, Partitioning, and Recovery of Enveloped Viruses in Untreated Municipal Wastewater

Quantitative analysis of SARS-CoV-2 RNA from wastewater solids in communities with low COVID-19 incidence and prevalence

This manuscript is one of its kind where the virus extraction is from wastewater solids and can fill many research gaps in WBE of SARS-CoV-2

An Economical Kit-Free Method for Direct Capture of SARS-CoV-2 RNA from Wastewater

Applicability of polyethylene glycol precipitation followed by acid guanidinium thiocyanate-phenol-chloroform extraction for the detection of SARS-CoV-2 RNA from municipal wastewater, Science of The Total Environment

Detection and quantification of SARS-CoV-2 RNA in wastewater and treated effluents: Surveillance of COVID-19 epidemic in the United Arab Emirates

Benchmarking virus concentration methods for quantification of SARS-CoV-2 in raw wastewater

Preliminary results of SARS-CoV-2 detection in sewerage system in Niterói municipality

Early-pandemic wastewater surveillance of SARS-CoV-2 in Southern Nevada: Methodology, occurrence, and incidence/prevalence considerations

Surveillance of SARS-CoV-2 RNA in wastewater: Methods optimization and quality control are crucial for generating reliable public health information

This paper discusses the optimization techniques while performing WBE of SARS-CoV-2

SARS-CoV-2 in Detroit Wastewater

Implementation of environmental surveillance for SARS-CoV-2 virus to support public health decisions: Opportunities and challenges, Current Opinion in Environmental Science & Health

This is a crucial manuscript for the present review paper. It discusses about WBE implementation

PCR inhibitors -occurrence, properties and removal

Surveillance of enteric viruses and coliphages in a tropical urban catchment

Effects of rainfall events on the occurrence and detection efficiency of viruses in river water impacted by combined sewer overflows, Science of The Total Environment

This paper discusses the flow normalization and is an important base for the present review

Microfluidic Quantitative PCR for Simultaneous Quantification of Multiple Viruses in Environmental Water Samples

Validation of Internal Controls for Extraction and Amplification of Nucleic Acids from Enteric Viruses in Water Samples

This paper discusses about the proper utilization of internal/external controls during virus detection

Mechanisms of Pathogenic Virus Removal in a Full-Scale Membrane Bioreactor

Norovirus and FRNA bacteriophage determined by RT-qPCR and infectious FRNA bacteriophage in wastewater and oysters

Evaluation of pepper mild mottle virus, human picobirnavirus and Torque teno virus as indicators of fecal contamination in river water

Occurrence of Viruses and Protozoa in Drinking Water Sources of Japan and Their Relationship to Indicator Microorganisms

RT-qPCR assays for SARS-CoV-2 variants of concern in wastewater reveals compromised vaccination-induced immunity

Direct RT-qPCR assay for SARS-CoV-2 variants of concern (Alpha, B.1.1.7 and Beta, B.1.351) detection and quantification in wastewater

ddPCR: a more accurate tool for SARS-CoV-2 detection in low viral load specimens

Wastewater-based epidemiology biomarkers: Past, present and future

Co-quantification of crAssphage increases confidence in wastewaterbased epidemiology for SARS-CoV-2 in low prevalence areas

Organic micropollutants discharged by combined sewer overflows -Characterisation of pollutant sources and stormwater-related processes

Data filtering methods for SARS-CoV-2 wastewater surveillance

Tools for interpretation of wastewater SARS-CoV-2 temporal and spatial trends demonstrated with data collected in the

This paper discusses about how to interpret WBE data and correlate it with clinically tested cases

Coronavirus resource center

COVID-19 surveillance in Southeastern Virginia using wastewaterbased epidemiology

Implementing building-level SARS-CoV-2 wastewater surveillance on a university campus

Wastewater surveillance of SARS-CoV-2 across 40 U.S. states from

Potential Sensitivity of Wastewater Monitoring for SARS-CoV-2: Comparison with Norovirus Cases

This paper critically discusses about the sensitivity of WBE of SARS-CoV-2 as compared to other viruses

SARS-CoV-2 wastewater surveillance data can predict hospitalizations and ICU admissions

Norovirus Dynamics in Wastewater Discharges and in the Recipient Drinking Water Source: Long-Term Monitoring and Hydrodynamic Modeling

Identification of environmental determinants for spatio-temporal patterns of norovirus outbreaks in Korea using a geographic information system and binary response models, Science of The Total Environment

This is an important manuscript that discusses on viruses and protozoa in drinking water and how they are source tracked.[