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com Current Opinion in

ScienceDirect Environmental Science & Health

Sewage surveillance for SARS-CoV-2: Molecular detection,

quantification, and normalization factors
Payal Mazumder1, Siddhant Dash2, Ryo Honda3,
Christian Sonne4,5,6 and Manish Kumar1
6
Jiangsu Co-Innovation Center of Efficient Processing and Utilization
Abstract
of Forest Resources, International Innovation Center for Forest
The presence of severe acute respiratory syndrome corona- Chemicals and Materials, College of Materials Science and Engi-
virus–2 (SARS-CoV-2) in wastewater systems provides a pri- neering, Nanjing Forestry University, Nanjing, Jiangsu, 210037, China
mary indication of the coronavirus disease 2019 (COVID-19)
spread throughout communities worldwide. Droplet digital po- Corresponding authors: Mazumder, Payal (payal.spinnersend@gmail.
com); Kumar, Manish (manish.env@gmail.com)
lymerase chain reaction (dd-PCR) or reverse transcription-
polymerase chain reaction (RT-PCR) administration of SARS-
CoV-2 in wastewaters provides a reliable and efficient tech- Current Opinion in Environmental Science & Health 2022,
nology for gathering secondary local-level public health data. 28:100363
Often the accuracy of prevalence estimation is hampered by This review comes from a themed issue on COVID-19 in environment:
many methodological issues connected with wastewater sur- Treatment, Infectivity, Monitoring, Estimation
veillance. Still, more studies are needed to use and create Edited by Manish Kumar, Ryo Honda, Prosun Bhattacharya, Dan
efficient approaches for deciphering the actual SARS-CoV-2 Snow and Payal Mazumder
indication from noise in the specimens/samples. Nearly For a complete overview see the Issue and the Editorial
39–65% of positive patients and asymptomatic carriers expel
https://doi.org/10.1016/j.coesh.2022.100363
the virus through their faeces however, only ~6% of the
2468-5844/© 2022 Elsevier B.V. All rights reserved.
infected hosts eject it through their urine. COVID-19 positive
patients can shed the remnants of the SARS-CoV-2 RNA virus
within the concentrations ~103 –108 copies/L. However, it can Keywords
decrease up to 102 copies/L in wastewaters due to dilution. RT-PCR, dd-PCR, SARS-CoV-1, Wastewater-based surveil-
lance, Epidemiology.
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 world-
wide. Virus extraction or recovery from the wastewater (How-
Introduction
As of November 2021, more than 262 million
ever, due to lack of knowledge, established procedures, and
confirmed cases of COVID-19 have appeared world-
integrated quality assurance/quality control (QA/QC) ap-
wide, triggering the ongoing pandemic. Wastewater-
proaches, the novel coronavirus RNA investigation for esti-
based epidemiology (WBE) has proved to be a swift
mating current illnesses and predicting future outbreaks is
and supplementary tool for health authorities world-
insufficient and/or conducted inadequately. The present
wide to monitor COVID-19 and also track the newly
manuscript is a technical review of the various methods and
emerging variants of concern. The use of WBE has
factors considered during the identification of SARS-CoV-2
previously been effective in monitoring a range of
genetic material in wastewaters and/or sludge, including tips
harmful viruses including polio, Noro, dengue [1], and
and tricks to be taken care of during sampling, virus concen-
recently SARS-CoV throughout the world [2e4]. It
tration, normalization, PCR inhibition, and trend line smooth-
targets the residues of the viruses’ DNA/RNA that
ening when compared with clinically active/positive cases.
function as the pathogens’ population quantitation.
Addresses Unlike other respiratory viruses, the SARS-CoV-2 virus
1
Sustainability Cluster, School of Engineering, University of Petroleum is located in most infested people’s gastrointestinal
and Energy Studies, Dehradun, Uttarakhand, 248007, India tracts and faeces [5]. Its injection into the wastewater
2
Department of Civil Engineering, Indian Institute of Technology
Guwahati, Guwahati, Assam, 781039, India
streams was found in the initial pandemic phases,
3
School of Geosciences and Civil Engineering, Kanazawa University, even prior to the first case in the communities were
Kakumamachi, Kanazawa, Ishikawa, 920-1192, Japan discovered [6]. WBE, therefore, holds the capability of
4
Department of Ecoscience, Aarhus University, Roskilde, DK-4000, acting as an early warning system for the development
Denmark of COVID-19 at a societal stage by analyzing the sig-
5
Henan Province Engineering Research Center for Biomass Value-
Added Products, Henan Agricultural University, Zhengzhou, Henan,
nals of the viral load in wastewater samples pooled by
450002, China population [7].

www.sciencedirect.com Current Opinion in Environmental Science & Health 2022, 28:100363

2 COVID-19 in environment: Treatment, Infectivity, Monitoring, Estimation
It is worth noting that changes in wastewater SARS-
CoV-2 RNA content are not necessarily proportionate
to changes in confirmed cases or incidences [8e11].
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 move-
ment. 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, the positive
ratio decreases when examinee is selected randomly or
an examinee group becomes larger). These fluctuations
in confirmed cases also cause a gap between confirmed
cases and wastewater epidemic data, as well as catch-
ment features and experimental errors. Therefore, more
profound knowledge is needed about the SARS-CoV-2
virus variability in wastewaters and how that corre-
sponds to the actual occurrence or dominance of
COVID-19 in the conducive population to ensure rele-
vance of the COVID-19 WBE for public health policy-
making. 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 inconsis-
tency in wastewaters [12e14].
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 vari-
ability, 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 faecal
material induced by dilution, primarily using the two
most familiar human faecal viral indicators (i.e., cross-
assembly phage (crAssphage) and pepper mild mottle
virus (PMMoV)) [16]. There are several limiting con-
ditions in the detection of the novel coronavirus in
wastewater systems. Municipal wastewater is a compli-
cated 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 minuscule 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
Current Opinion in Environmental Science & Health 2022, 28:100363
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.
Virus extraction from wastewater sample(s)
To get detectable levels of viral nucleic acid, a 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 [20e22] as well as time or flow propor-
tional composite samples [22e24]. 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 dis-
tance 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 fre-
quency is little and/or the sampled population size is
small, such as in near-source 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 coro-
navirus monitoring, in wastewater [25e29]. The ma-
jority 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’ depend-
ability, 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 con-
centrations in wastewater (used for process control)
[32]. Two more enveloped coronaviruses (BCoV
and OC43) are employed further to understand the ef-
ficacy in detecting SARS-CoV-2 in wastewater of which,
the concentrating pipette (CP)-based concentration
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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 two times greater with the CP 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 CP approach, since
enveloped viruses have been discovered as increasingly
connected to the surface of the particles present in
wastewater compared to viruses that lack envelopes, in
prior research [33]. Virus recovery efficiency post frac-
tionation, polyethylene glycol (PEG) precipitation, and
viral RNA extraction in vesicular stomatitis virus (VSV)-
spiked wastewater samples were done by Ref. [34]
(Equation (1)).
Total gene copies of VSV recovered
Viral recovery efficiencyð%Þ ¼
Total VSV gene copies spiked ingrit=sludge
[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 h. This
approach helps researchers at the University of Cali-
fornia, 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 precipi-
tation [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 Ref. [42], in which
the amount of RNA of SARS-CoV-2 conducted from
wastewater of 45 L quantity via electropositive car-
tridges, is the only large-volume concentration tech-
nique published in previous articles; nonetheless, being
very labour-intensive. Table 1 shows the various ap-
proaches by researchers reported recently on the
wastewater monitoring methods of SARS-CoV-2.
PCR types/techniques for detecting and
quantifying SARS-CoV-2
In wastewater matrix/systems, detection of SARS-CoV-2
RNA starts with sampling from inside a wastewater
network structure such as 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/
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Wastewater surveillance of SARS-CoV-2 Mazumder et al. 3
or RT-qPCR (centred on a standard plot) for identifi-
cation and quantification of SARS-CoV-2 genetic ma-
terial. Poor viral retrieval and/or testing of minor
effective sample volume (ESV), inefficiency of extract-
ing 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 [47e49], which are added before
nucleic acid extractions; and (3) RT-qPCR controls
 100% (1)
[50e52], 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 the
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].
dd-PCR is considerably vigorous in managing PCR in-
hibition conditions than standard quantitative RT-
qPCR assays. The Qiagen kit operated for extracting
RNA contains many inhibitors elimination processes
[53]. SARS-CoV-2 variants can be detected via PCR
and/or Next-generation sequencing (NGS) technique
[54,55]. designed direct qPCR probes and primers on
the basis of variations in sequences between the newly
emerging strains (Alpha (B.1.1.7), Beta (B.1.351),
Gamma variant (P.1), and Delta variant (B.1.617) with
the wild type SARS-CoV-2. The novel primers for
detecting P.1 and B.1.617 variants had shown to be both
specific and sensitive, with a limit of detection (LOD)
10
in both sterile conditions and sewage environment
systems. Although dd-PCR has not been extensively
used, it has been evaluated and proved helpful, mainly
when viral loads are low, as they are during the decline in
the virus load and/or in the course of early phases of virus
dissemination [56]. As a result, more research is needed
to determine the efficacy of RT-ddPCR.
Current Opinion in Environmental Science & Health 2022, 28:100363
Current Opinion in Environmental Science & Health 2022, 28:100363

4 COVID-19 in environment: Treatment, Infectivity, Monitoring, Estimation

Table 1

Recent studies on SARS-CoV-2 sampling, sample pre-treatment, RNA gene detection, and sequencing for COVID-19 surveillance via non-clinical approach.

sample Region Population Sampling type Time period Virus filtration and Recovery Detection Internal control/ Sequencing/ Reference
size concentration efficiency/ method and PCR inhibition variant analysis
Normalization target gene(s) (+/−)
biomarkers

Pumping station Southeast 42,612–1, Grab sampling January 2020 Direct RNA – RT-qPCR, Oncorhynchus + (Sanger and Ahmed et al.,
(1) and Queensland, 106,892 and –April 2020 extraction from keta MiSeq, 2020a [15]
Wastewater Australia automated electronegative (O. keta)- Illumina)
treatment sampling membranes and copy number
plants (2) (conventional ultrafiltration 104/reaction
refrigerated
autosampler
and in-situ
high-
frequency
autosampler)
Wastewater Central Ohio, 14,000–49, 24-h composite July 2020 Adsorption- Spiking surrogates: dd-PCR, N- Firefly + (Next Ai et al., 2021
treatment US 000 samples-twice –January precipitation by male-specific gene and (Coleoptera) generation [8]
plants (9) a week 2021 a positively coliphage MS2 E-gene Luciferase sequencing)
charged filter (ATCC cat. No. control RNA
followed by 15597-B1),
flocculation and bovine
centrifugal coronavirus
ultrafiltration (BCoV strain
ML-6 mebus),
and human
coronavirus
OC43 (ATCC
cat. No. VR-
1558)
Wastewater California–San 82,818–1, 24-h time- April 2020 Modified 4S crAssphage RT-qPCR, N1 VetMAX™ – Greenwald
treatment Francisco 500,000 weighted –September method CPQ_056, gene Xeno™ et al., 2021
facilities (6) Bay area, US composite 2020 pepper mild Internal [61*]
samples- mottle virus coat positive
weekly protein gene control
(PMMoV), (Xeno)
Bacteroides 16S
ribosomal RNA
HF183/
BacR287,
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bovine
coronavirus
transmembrane
protein gene
(BCoV),
Synthetic
Oligomer
Construct T33-
www.sciencedirect.com

21 free-RNA
(SOC), and
human 18S
rRNA
Wastewater Ishikawa and 31,501 - Grab sampling March 2020 PEG precipitation – qRT-PCR, Murine + (Sanger Hata et al.,
treatment Toyama 233, –May 2020 and CDCN2, norovirus sequencing) 2021
plants (5) Prefecture, 480 quantification of CDCN3,
Japan F phage and NIID
assays
Wastewater Amsterdam 267,900 - 24 h composite March 2020 Centrifugation and CrAssphage RT-qPCR Mouse + (Cell culture Heijnen et al.,
treatment and Utrecht, 669, sampling –March 2021 ultrafiltration CPQ_064 and RT- Hepatitis and whole 2021
plants Netherlands 400 ddPCR, N2 Virus (MHV)- genome
assay A59 sequencing)
Wastewater Ahmedabad, 7,800,000 Grab sampling May 2020 PEG precipitation – RT-qPCR, Bacteriophage – Kumar et al.,
treatment plant Gujarat, India ORF1ab, N MS2 2020 [17]
(1) and S gene
Wastewater Milan, Italy 900,000–1, 24 h composite February 2020 PEG-dextran – Nested RT- OneStep PCR + La Rosa
treatment 050,000 sampling –April 2020 method PCR and Inhibitor et al., 2020
plants (3) RT-qPCR, Removal Kit [3*]
ORF1ab
gene
Lift stations (7)Vancouver, 87,89,211 24 h composite February 2021 Magna Pure 96 – RT-qPCR, N- – + (Sdel and Peterson
and Edmonton, sampling –March 2021 DNA and Viral gene, and SN501Y et al., 2021
Wastewater Toronto, NA Large S gene assays)
treatment Montreal, Volume Kit
plants (15) Halifax, and
Northwest
Current Opinion in Environmental Science & Health 2022, 28:100363

Territories of
Canada

Wastewater surveillance of SARS-CoV-2 Mazumder et al.

Wastewater Milano and 200,0000 Grab sampling April 2020 Whatman GF/F Caffeine RT-qPCR, N, QIAMP Viral + (Ion Torrent Rimoldi et al.,
treatment Monza e and quantification ORF1ab RNA mini kit PGM 2020
plants (3)- raw Brianza, Italy nitrocellulose and E gene sequencer)
and treated Millipore MCE
wastewater filters
Manholes and Israel 92,406– 24 h composite June 23, 2021 MCE electro- – RT-qPCR, N- MS2 phage + (RT-qPCR Yaniv et al.,
Wastewater 644, sewage negative gene primer 2021 [54]
treatment plant 000 sample membrane (direct RNA probes)
extraction-
Zymo
Research
R2042
protocol)

5
6 COVID-19 in environment: Treatment, Infectivity, Monitoring, Estimation
Normalization
Normalization is recently considered effective since
viral concentration in wastewater is affected by dilution
with non-faecal wastewater and quantity of faecal
discharge at the corresponding time of sampling.
Normalization concepts using biomarkers, higher fre-
quency of sampling, sample replicate processing, and
smoothening/forecasting have been used to address
some of the drivers of variability in SARS-CoV-2 iden-
tification. 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, stan-
dardized concentrations of wastewaters to population
and flow rate across WBE experiments (e.g., caffeine)
are used [34]. COVID-19 WBE assays typically rely on
the detection of a non-enveloped plant virus viz.,
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
Figure 1
Wastewater sampling, virus concentration, internal controls, normalization of SARS-CoV-2 signal and variant detection for efficient public health-related
decision-making, and evaluation of vaccination efficiency against SARS-CoV-2 variants.
Current Opinion in Environmental Science & Health 2022, 28:100363
rRNA gene of the human-specific HF183 Bacteroides is
commonly utilized to identify faecal 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]. Figure 1 shows the various steps for
sample collection, virus concentration, PCR standards,
normalization of data, and sequencing for variant iden-
tification, all of which need to be taken extreme care of
for WBE of SARS-CoV-2. Moreover, flow normalization
method (with electrical conductivity (EC) check) is
economic and reliable [59] as shown in equations (2)
and (3).
Viral loading ¼ Virus concentration  Flow rate of wastewater
(2) Or
Viral loading per capita ¼ Virus concentration
Flow rate
 (3)
Population coverage
Also, the number of people in a catchment can be back-
calculated with the help of tracing biomarkers [60].
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C bm  Q
P¼ (4)
fbm
where, P = population/number of people in the watershed,
Cbm = concentration of the biomarker used (g/L), Q = flow
volume (L/d), and fbm = specific biomarker load (g/P/d).
Also, three equations to find the percentage of domestic
wastewater using flow data (equation (5)), EC data
(equation (6)), and crAssphage data (equation (7)) are
given as follows:
Inhabitants  DDWF
%Domestic sewageðQÞ ¼  100% (5)
V24h
Inhabitants  DDWF ECSample
%Domestic sewageðECÞ ¼ 
VDWFref ECDWF ref
 100%
(6)
Inhabitants  DWF CrAssSample
%Domestic sewageðCrAssÞ ¼  plot smoothing, which may be employed to lesser sam-
VDWF ref CrAssDWF ref
 100%
(7)
where, DDWF = average daily domestic wastewater gen-
eration by each person, V24h = wastewater volume
measured over 24 h sampling time, VDWF ref = average
wastewater volume during dry weather flow (40th percen-
tile of daily 261 volumes derived from 1-year time series of
flow monitoring), ECSample = measured EC values in sam-
ples, ECDWF ref = average EC of all samples whose flow
ranges between the 10th and 50th percentile of daily vol-
umes derived from 1-year time series of flow monitoring,
CrAssSample = genome copies of the phage/ml sample, and
CrAssDWF ref = average CrAss of all samples with flows
ranging between 10th and 50th percentile of daily volumes
derived from a 1-year time series of flow monitoring
(genome copies/ml sample).
[8] conducted wastewater surveillance of SARS-CoV-2,
where the measurements were taken from the influent
wastewater samples of the capital and seven other cities
of varying sizes in central Ohio for three target genes
(i.e., E gene regions, N1 gene, and N2 gene) in a period
ranging from July 2020 to January 2021. Quantification
of crAssphage and PMMoV (human faecal viruses) was
carried out, normalizing the detected SARS-CoV-2 gene
concentrations in the sample to regulate human-specific
faecal strength better. This followed normalization of
the RNA intensities using PMMoV RNA (concentration
(average) throughout the samples. Visually, both
methods had increased the concordance of viral con-
centrations and case numbers but not statistically
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Wastewater surveillance of SARS-CoV-2 Mazumder et al. 7
(Spearman rank correlation). Therefore, interestingly
neither the PMMoV nor the crAssphage normalization
procedures significantly improved the association with
new case numbers or the estimate models. In yet
another research carried out by Ref. [61], four normali-
zation indicators were used to account for variance in
wastewater sample faecal content viz., PMMoV, crAss-
phage, 18S rRNA (human), and Bacteroides ribosomal
RNA. CrAssphage had the slightest geographical and
temporal changeability of the group. Both normalized
to crAssphage and unnormalized data signals of
SARS-CoV-2 RNA peaks conclusively and notably
correlated (Kendall’s Tau-b (t) = 0.43 and 0.38,
respectively) with the clinical tested confirmed
COVID-19 case data trends.
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 are frequently utilized to examine real-time
clinical data patterns [62], low frequency of waste-
water sample collection (e.g., once or twice a week)
makes it difficult to use such an approach. As a result,
smoothing approaches such as locally weighted scatter
pling 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 band-
width selection technique is rarely mentioned [31,64].
The detection boundary is determined by the ap-
proaches employed to collect hereditary matter and the
scope of local clinical examining, and it might necessi-
tate sewer shed-specific evaluation. With clinical testing
capabilities, this value may change over time. A
methodical technique to estimate this amount across
researches can help understand non-detects and predict
the per capita quantum of SARS-CoV-2 positive cases
over which WBE (of SARS-CoV-2) becomes an effective
public health monitoring tool [61]. Recent research
used a dataset of 1687 samples to establish an evident
WBE case detection boundary that was huge enough 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 quan-
tify [66].
Future implications for the WBE tool
Over a dozen investigations worldwide demonstrate a
shift toward using wastewater inspection to estimate
SARS-CoV-2 transmission in different locations. SARS-
CoV-2 identification studies mostly show the slope of
the graph plotted between the cumulative infections’
cases and the number of copies of genes, post normali-
zation in the population living in the catchment area.
Current Opinion in Environmental Science & Health 2022, 28:100363
8 COVID-19 in environment: Treatment, Infectivity, Monitoring, Estimation
This slope can be linked to the number of new cases on
average over the sample period. As a result of the asso-
ciation, qPCR data may forecast future contagions.
Because wastewater sampling captures all infections,
whether symptomatic or not, a lag time enhanced the
association between the average number of new cases
and the slope. SARS-CoV-2 variants (Alpha (B.1.1.7),
Beta (B.1.351), Gamma variant (P.1), and Delta variant
(B.1.617)) can also be detected via wastewater surveil-
lance, and the emergence of new variants can be re-
ported while mitigation strategies for future outbreaks
in targeted regions can be focused.
[55] created a RT-qPCR technique for the fast, sensi-
tive, and direct identification of SARS-CoV-2 P.1 and
B.1.617 variants. WBE can be a valuable instrument for
explaining population diversity, immune response, and
monitoring and revealing vaccination attempts and dy-
namics of individual resilience to SARS-CoV-2 infection
in the community. Wastewater should play a major part
in the surveillance of a variety of different infectious
illnesses in the future [67]. developed connections
linking SARS-CoV-2 genetic material load in wastewater
and pandemic health indices using modelling ap-
proaches such as distributed/fixed lag modelling, linear
regression, and artificial neural networks. Such models
[68,69] can be very helpful in conducting risk assess-
ments for future outbreaks.
Conclusion
Both methodological studies for testing SARS-CoV-2 in
wastewaters and the effectuation of WBE happened
simultaneously during the COVID-19 epidemic.
Studies show that patterns in wastewater monitoring
figures match COVID-19 incidence trends; they also
offer strategies for making wastewater signals more
interpretable and comparable between studies. Clinical
and environmental monitoring data may be integrated to
construct robust models that can be used to examine the
ongoing COVID-19 infection dynamics and give an early
warning system for increasing hospital admissions. To be
relevant for decision-making for public health, COVID-
19 WBE must be sure that the resultant signal of SARS-
CoV-2 accurately represents the trends of COVID-19 in
the causative population. Data such as rigorous QA/QC
courses, characteristic sampling tactics, efficacious virus
concentration techniques and effectual RNA elution
and recovery methods, accurate assessment of PCR in-
hibition, the incorporation of controls for processing of
wastewater/sludge samples, significances for dd-PCR/
RT-PCR assay availability, selection and scientific eval-
uation of signal/trend, etc., are all recommended to
reduce incorrect positive and negative cases in SARS-
CoV-2 surveillance.
Current Opinion in Environmental Science & Health 2022, 28:100363
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could
have appeared to influence the work reported in
this paper.
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