An opinion on Wastewater-Based Epidemiological Monitoring (WBEM) with Clinical Diagnostic Test (CDT) for detecting high-prevalence areas of community COVID-19 Infections

Abstract

Wastewater-Based Epidemiological Monitoring (WBEM) is an efficient surveillance tool during the COVID-19 pandemic as it meets all requirements of a complete monitoring system including early warning, tracking the current trend, prevalence of the disease, detection of genetic diversity as well asthe up-surging SARS-CoV-2 new variants with mutations from the wastewater samples. Subsequently, Clinical Diagnostic Test is widely acknowledged as the global gold standard method for disease monitoring, despite several drawbacks such as high diagnosis cost, reporting bias, and the difficulty of tracking asymptomatic patients (silent spreaders of the COVID-19 infection who manifest nosymptoms of the disease). In this current reviewand opinion-based study, we first propose a combined approach) for detecting COVID-19 infection in communities using wastewater and clinical sample testing, which may be feasible and effective as an emerging public health tool for the long-term nationwide surveillance system. The viral concentrations in wastewater samples can be used as indicatorsto monitor ongoing SARS-CoV-2 trends, predict asymptomatic carriers, and detect COVID-19 hotspot areas, while clinical sampleshelp in detecting mostlysymptomaticindividuals for isolating positive cases in communities and validate WBEM protocol for mass vaccination including booster doses for COVID-19.

Graphical abstract

1. Introduction

The current ongoing coronavirus disease-2019 (COVID-19) having flu-like symptoms caused by a causative agent,severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has wreaked havoc, on global economics, businesses,communities, and public health due to widespread infection, with 43.7 million confirmed cases and1.17 million deaths in 218 countries as of March 31, 2022 (WH0 2022; JHU 2022) (1). Two major obstaclesto managingSARS-CoV-2 rapid infections aredifficulties identifyinginfected people based on their signs and symptoms as well as preventingthe spread of viral infections in the communities(2). Over the past 18 months, the SARS-CoV-2 virus has diversified through multiple new mutationsand various genetic variants such as Alpha (B.1.1.7) with seven, Beta (B.1.351) with nine, Gamma (P.1) with 12, Delta (B.1.6, B.1.6.2) with 17 new mutations in spike protein gene, the latestOmicron (B.1.1.529) and Neocov variants, have been discovered and propagated into different parts of the world as variants of interest to concern(3, 4, 5). These altered genetic factors increased transmissibility, virulence, disease severity, and mortalities while they also decreased the effectiveness of current therapeutics and vaccines (6, 7, 8).

Since the beginning of the COVID-19 pandemic, the standard clinicaldiagnostic testing (CDT) is a recognized, valid system for monitoring infectious diseaseslike COVID-19 with some negative sides (9), which relies on the patient's signs and symptoms. As an alternative to individual tracking,wastewater-based epidemiology (WBE) has been used on a wide scale across theworld (10∗, 11∗, 12) to monitor the prevalence of COVID-19 patients. Despite the simplicity of wastewater sampling and transportation on time, viral RNA concentration and extraction are very difficult for low RNA quantities (13,14). Hence, it is verycrucial to establish a unified system incorporating CDT and WBEM to identify infectedindividuals with COVID-19 hotspots while discovering new variants and mutations, monitoringthe current pandemic scenario, followed by anticipating future waves (15). CDT and WBEM can be used synergistically to track localCOVID-19 epidemics where clinical samples will be used to identify the SARS-CoV-2 symptomatic patients and WBEM will be optimizedas a validated method for further analyses of wastewater released into communal drains from individual household drains and public places (e.g. bus and rail stands, airports, rivers, and market) (16∗, 17∗, 18∗, 19∗, 20). As a result, adapting the collective approach combiningWBEM and CDT could relieve burdens on the public health system, while it assists in making informed decisions for better and proper timely treatments, receiving vaccines, or booster dosages of vaccines for COVID-19.

.Based on our experience, it is worth mentioning that continuing WBEM without proper sanitation systems is strenuous, especially for low-middle income countries or non-WASH (Water, Sanitation and Hygiene) countries (1,21,22). However, adapting a combined approach can be the best model for both the developing and developed world (25).

In developing countries, WBEM of COVID-19 is more challenging without CDT, as the majority of households are not connected to sewerage systems. The CDT for SARS-CoV-2 can detect the viral genetic markers of the viral RNA in-between 7–14 days following the exposure and are unable to detect asymptomatic individuals (silent spreaders of COVID-19) within the communities (26,27). WBEM is an approach for tracking the pandemic through the identification of severely infected areas (COVID-19 hotspot zone) and monitoring of the infection trends (24,27). However, the recovery of the genetic biomarkers of the SARS-CoV-2 viral RNA in wastewater is very challenging due to differential stability in sewage streams, various environmental factors such as rainfall and temperature, as well as the presence of inhibitory substances (Ribonuclease Enzyme-RNase) (29,30). As a result performing a well-structured surveillance system combining both CDT and WBEM for symptomatic, asymptomatic, and paucisymptomatic carriers with unusual mutations as well asidentifying early warning for new variants are also helpful in vaccine development. From our recent completed 30 days follow-up on the quantitative analyses of SARS-CoV-2 genetic materials in wastewater from the residence of a positive patient family (Islam et al., 2022 under revision STOTEN-D-22-08275R1), SARS-CoV-2 positive patient number was identified as the lowest when the CT value was observed the highest (lowest copy number) in wastewater samples. On the other hand, the number of positive patients was found the highest with the corresponding lowest CT value (the highest copy number) in sewage samples as detected in the same study. In addition, increased signals of the SARS-CoV-2 genetic materials were noticed earlier in WS compared to positive patient viral load in clinical samples.

There are limited studies that link the concentation of SARS-CoV-2 viral biomarkers in wastewater with the identification of clinical cases in a specific residential area lacking wastewater treatment plants in developing countries (31, 32, 33). The combined CDT and WBEM follow-up study was performed in our laboratory to determine the relationship between the positive cases of SARS-CoV-2 infections and their discharged wastewater viral loads from one single house enrolling the entire family members’ clinical sample in a developing country without having a proper sewage system. The research findings demonstrated that a wastewater sample monitoring system tailored to a specific location could be established as a tool to identify SARS-CoV-2 infection and complement the clinically testing. This review emphasizes the combined monitoring of SARS-CoV-2 using CDT and WBE systems can guide the way forward for effective surveillance of the prevalence of the infectious disease such as COVID-19.

2. Clinical Diagnostic Test (CDT) and Wastewater-Based Epidemiologic Monitoring (WBEM)

The accurate and rapid clinicaldiagnostic tests are essential for identifying the SARS-CoV-2 positive cases, contact tracing, and making public health decisions (9). Clinical testing is a conventional method for monitoring the status of COVID-19, notably, the clinical signs and symptoms of a patient such as fever, dry cough, headache, and shortness of breath usually develop 2-14 days after the exposureto SARS-CoV-2 (5,25) (Supplementary Table ST 1). The CDT is recommended for the diagnosis of any diseases based on the patient's specific signs and symptoms (35) (Figure 1 , Supplementary Figure SF1). However, the maximum COVID-19 positive individuals are asymptomatic (36,37), and clinical data may be limited due to testing capacity, reagent costs, laboratory facilities with proper instruments, expert hands, and availability issues.

Figure 1.

COVID-19 patients’ clinical diagnostic tests include general clinical signs and symptoms, imaging findings, and laboratory markers (38). In vitro, diagnostic, and clinical laboratory tests include molecular techniques e.g., viral antigen detection, antibody tests; nucleic acid amplification tests (NAAT); real-time polymerase chain reaction test (RT-PCR); next-generation sequencing (NGS); cell culture; enzyme-linked immunosorbent assay (ELISA); and traditional clinical tests: neutrophil-lymphocyte ratio (NLR); c-reactive protein test (CRP); erythrocyte sedimentation rate test (ESR,); IL-6/interleukin-6; lactate dehydrogenase test (LDH); aspartate aminotransferase test (AST); alanine aminotransferase test (ALT);imaging method: computed tomography (CT); ultrasound sonography (USG), X-ray.

The specific catchment area or community wastewater can be used to identify and observe COVID-19 infection scenarios in the same area, in the same manner, that was previously used for eradication of poliovirus, and this is recognized as wastewater-based epidemiology (WBE) (39). The SARS-CoV-2 genetic materials havebeen identified in feces from pre-symptomatic persons even1–5 days before the positive clinical test(29, 30) and in people with mild signs and symptoms (41). Recent investigationsof a few WBE studies have detected COVID-19 patients before the onset of clinical symptoms from feces samples, and 48-67% of diseased people had SARS-CoV-2 viral RNA in their stool, which survives in wastewater and can last up to >33 days (41, 42, 43, 44). Previous findings showed anassociation between wastewater viral concentration and COVID-19 confirmed cases where SARS-CoV-2 viral RNA limit between 2.0 and 6.0 log₁₀ gc/L (genomic copies per liter), which is similar to our recent research findings (36, 38, 41). In addition, the accuracy of WBE was found in many studies (35, 36, 37, 38) where Betancourt et al. (2021c), confirmed the WBEM has a sensitivity of 76.0%, specificity of 90.7%, positive and negative predictive value of 79.8%, and 88.6%, respectively when findings of wastewater samples were compared with clinical samples (46). Furthermore, according to previous studies, SARS-CoV-2 RNA prevalence in stool was higher (48.1%) than in patients detected with gastrointestinal symptoms (17%) (42). Although WBEM is capable to detect SARS-CoV-2 RNA genetic biomarkers for monitoring the pandemic, there is an ongoing debate over how wastewater data should be used and to what extent the approaches are useful to public health decisions.

As the COVID-19 pandemic continues, individual clinical diagnostic testing (CDT) did not represent itself as a holistic approach to community health status determination. One major concern with COVID-19 pandemic is that most cases in the United States, patients were generally asymptomatic and pre-symptomatic, allowing infected people to spread the virus as healthy carriers (47). Moreover, a significant percentage of COVID-19 survivors might still be carrying and shedding the virus (48). Hence, in addition to the clinical test, wastewater surveillance should be used together with clinical data to infer the average virus shedding patterns at a population level (49,50). Figure 2 depicts a high-level overview of the WBEM system from sample collection to result in interpretations. The selection of WS collection sources playsan important role as a proper catchment area (wastewater treatment plants, sewer drains, primary networking system or communal watershed, river courses, bus stand, airports) (22). Heat treatment (60⁰C, 30 minutes), filtration (to remove large particles), or chemical treatment with NaOCl can be used for sample processing and disintegrating the viruses (19). Several methods are already used in various studies for concentrating viral biomarkers like polyethylene glycol (PEG), ultrafiltration, ultracentrifugation, centrifugation, or skim milk procedure (51). Viral nucleic acid can be extracted using manually (Trizol Based) or kit-based (Qiagen, Thermo Fisher) (52). For calculating viral copy number maximum studies have used equations 1 and 2.

Figure 2.

Overview of WBEM procedure. Wastewater sample collection and processing, viral RNA concentration, nucleic acid/RNA extraction from the concentrated samples, interpretation of RT-PCR results, and monitoring the trend of disease outbreak compared with CDT and Whole Genome Sequencing (WGS) for new variants and mutations.

Number of infected individuals = $\frac{\frac{\text{RNA}\text{Copies}}{\text{L}\text{Water}}\times \text{L}\frac{\text{Water}}{\text{day}}}{\frac{\text{g}\text{feces}}{\text{day}}\times \frac{\text{RNA}\text{Copies}}{\text{g}\text{feces}}}$ Equation 1 (51).

Number of infected individuals = $\frac{\text{No}.\text{of}\text{RNA}\text{Copies}\text{per}\text{liter}}{\text{Contribution}\text{of}\text{RNA}\text{Copies}\text{per}\text{person}\text{to}\text{total}\text{sewage}\text{water}}$ Equation 2 (53).

Positive, negative, no-template, and extraction control controls should be used with standard curve calculation as well as PCR inhibitors should check accordingly to MIQE rules(54). Major roles and drawbacks of both WBEM and CDT are given in Table 1 . Considering the previous research outcomes on WBEM and CDT activities with various validated methods and comparisons of the two approaches, the following future recommendations should be done for (1) disease burden correlation (2) trend analysis compared with clinical tests (3) observing the effectiveness of the intervention with the declining number of patients, and (4) detection of hotspot areas with COVID-19 cases for vaccination and booster doses (55).

Table 1.

A comparison of the major advantages and drawbacks of CDT and WBEM.

Parameters	Advantages	Disadvantages	Reference
CDT	•High sensitivity and specificity. •Rapid testing method. •Determination of new variants. •Simple, safe, and cost-effective. •Antigen and nucleic acid amplification methods are more suitable for the early and accurate detection of acute infection with COVID-19. •Antibody detection methods play an important role in seroprevalence analysis, allowing countries to estimate the rate of exposure and take. Precautionary measures to handle waves of the pandemic which are useful for epidemiologic purposes.	•Not applicable for asymptomatic patients. •Test kits are costly. •Cross-reactivity and false positive results. •The sensitivity of antibody tests for the detection of active infection is highly variable and not suitable for the early detection of COVID-19. •Limitation in handling pandemic situationsusing RT-PCR technique as the standard test. •Expensive equipment, well-trained staff, and equipped laboratories are required.	(59), (60),(61),(62), (63)
WBEM	•Cost-effective. •Useful to track COVID-19 hotspots, an estimate of the number of patients, and disease trends in communities. •Severe as an early warning tool, signaling the presence of infected people in a certain community or within a specified wastewater treatment plant (WWTP) catchment region. •Can be used to back-calculate chemical exposure or usage, as well as the prevalence of infection, with the use of appropriate models.	•Quantifying viruses and viral nucleic acids in wastewater are notoriously challenging. •Viral concentration in WS is influenced by rainfall, industrial inputs, substances that could degrade viruses (detergent, pH, salt), and the amount of feces. •RT-PCR inhibitors present in WS and chemicals used for concentration might inhibit the detection system. •Without a proper sewer facility, it is difficult to implement this tool.	(64), (65) (23), (66), (67)

One previous study in Massachusetts between March and May 2020 found similar trends of the SARS-CoV-2 virus in wastewater with the number of affected patients (56). Another study in Utah used 9-week wastewater sampling and found a link between a community outbreak and an increase in SARS-CoV-2 RNA (57). The SARS-CoV-2 virus concentrations in wastewater samples in Ottawa, Canada surged by more than 400% just 48 hours after a 300% or greater rise in recognized cases (58), and in Utah showed a strong link between community outbreaks and an increase in SARS-CoV-2 RNA in wastewater (57). Environmental parameters are also linked to SARS-CoV-2 genetic materials, as evidenced by an increase in wastewater temperature resulting from a decrease in viral gene copy numbers (2).

3. Examples of combined wastewater-based monitoring with clinical diagnostic tests

The previous WBS studies have found a direct correlation between CDT-confirmed COVID-19 cases and wastewater SARS-CoV-2 viral concentration (68,69). Various findings reflected how SARS-CoV-2 WBEM provided early warnings in the population analyzed, and detected viral RNA in WS before CDT (65,66,67). Viral RNA was found in wastewater samples in Milan, Italy few days after the first confirmed COVID-19 patient by clinical test (70), in Australia (Brisbane), when there were hundreds of clinical cases (71); in Japan (Yamanashi Prefecture), when clinical test results were at their peak (72); and in Spain (Murcia), when the COVID-19 cases were the least in the Iberian Peninsula (73) (Table 2 ). The detection of SARS-CoV-2 RNA in the wastewater treatment plant was also reported initially in Louisiana, USA (61), Gujarat, India (74), Dubai (75), Gothenburg, Sweden (76), and in the Southeast England of the United Kingdom (77). Medema et al. (2021) (78) successfully detected SARS-CoV-2 viral RNA from the city wastewater in the Netherlands six days before the first confirmed clinical case (79), and another group of researchers from northeastern of the United States reported that viral titers in WS indicated COVID-19 infections were higher than clinical reports (80).

Table 2.

Salient examples of integration of wastewater monitoring with clinical testing data.

Country	Area/Population/Time	Sampling Site, type	Concentration method	RNA/Nucleic Acid extraction Kit Name	RT-PCR Kit and Covered gene	Viral load range (gc/L)/CT value	Clinical cases/range	Main Findings (Correlation of Waste water result with clinical data)	Reference
Qatar	Doha 2.8 million January 2021	WWTP Influent Raw wastewater composite	PEG	Quick-RNA Viral Kit	Bio-Radi Taq Universal Probes One-Step Kit	7.8×103 to 5.4×105	31,181 to 542,313	A similar trend as both WS and CS were decreasing at the same time	(90))
Germany	North Rhine-Westphalia 3,725,633 August 2020		Filtration	RNA Blue Kit	N gene, S gene, and ORF1ab gene)	NF 6.16 × 1014	1-10000	WS could be used as an early warning tool.	(91)
USA	Utah 1597460 July 2021	Influent, activated and anaerobic ally digested sludge, composite	Filtration Centrifugation	AllPrep Power Viral DNA/RNA kit	TaqPathTM 1-Step RT-qPCR Master Mix N1, N2	1.0 × 105 to 1.0 × 106 GC/L.	NF	Primary sludge can be used to predict disease prevalence	(92)
USA	New Orleans 290,321 June 2020	3.1×103 and 4.3 ×103 copies/L	Ultrafiltration and adsorption-eluting using electronegative membrane	ZR Viral RNA Kit	0-7000	3.1 × 103–7.5 × 103/L	PerfecTa qPCR ToughMix (Quantabio, Beverly, MA)	Detection of viral RNA in WS after first caseby clinical diagnostic test.	(61)
Mexico	3.8 million	WWTP Grab	NF	RNeasy Mini Kit	QuantiNova® SYBR® Green PCR Kits (Qiagen, USA) N1, N2	1.9 ×103 to 3.5×106	74 to 82,690	WS detected SARS-CoV-2 2–7 days earlierthan clinical reports.	(93)
India	Ahmedabad NF September 2020	WWTP Grab Influent wastewater	PEG	NucleoSpin® RNA Virus isolation kit	TaqPathTM Covid-19 RT-PCR Kit ORF1ab, N, and S	(∼10,729 copies/L) > September (∼3047 copies/L) > October (∼454 copies/L)	50-650	Identification of COVID-19 hotspots	(94)
USA	Boston(Massachusetts) NR June, 2021	WWTP raw sewage composite	Pasteurization, Filtration	RNeasey PowerSoil Total RNA Kit,	Amicon Ultra-15 centrifugal ultrafiltration units (Millipore UFC903096)	Log(10+1)(4, 5, 6)/l 200000-1200000	NF N1, N2	WS detected SARS-CoV-2 RNA before CS in the first wave.	(95)
France	Nancy 250,000 April2020	raw WWTP	NucliSENS® lysis buffer	One-Step RT-ddPCR™ Kit for Probes	Two concentration procedures, based on ultrafiltration and on PEG 6000 precipitation, r	2.1 × 107 ± 1.1 × 107 gc/L and 1.6 × 107 ± 1.4 × 107 gc/L uncon and conc	100-1000	Decrease viral load during lockdown with decreasing patient's number of COVID-19.	(96)
UK	Gwynedd, Cardiff,Liverpool,Manchester, Wirral, Wrexham ∼3 million July 2020	Untreated WWTP Influent; Composite and Grab samples	Centrifugation,Ultrafiltration	NucliSENSeasyMag	Ultrasense Reaction Mix,Enzyme Mix; N1 and E	<1.2×103-1.5×104/100mL	5000-20,000	Both positive and negative correlation	(83)
UK	South East England 4 million April 2020	Raw WWTP influent; Composite	Filtration	High Pure Viral RNA kit	Invitrogen SuperScript III One-Step RT-PCR System	3.5-5.27 log 10gc/l 3.1×103 -6.0× 105	0-10,000	Able to detect prevalence variant by WS sequencing	(97)
Netherlands	Amsterdam, DenHaag, Utrect, Apeldoorn, Amersfoot, Tilburg and Schiphol 2,802,800 July 2020	WWTP Composite	Ultrafiltration	Biomerieux Nuclisens kit	Neasy PowerMicrobiome Kit (Qiagen, Hilden, Germany)	2.6−30 gene copies per mL	20-140	WS detected RNA prior to CS.	(98)
South Africa	NF Durban April 2021	WWTP composite and Grab Raw samples	Ultrafiltration, Adsorption	RNeaseyPowerSoil Total RNA Kit,	Primer, Probe	1.55×105-7.32×106	95,000 to 2.3 million	WS viral load similar with CS	(99)
Australia	Brisbane 934,000 November,2020	WWTP Composite	Filtration	RNeasy Mini Kit	iTaq™ UniversalProbesOne-Step Reaction Mix N1, N2, E	135 to 11,992 gene copies (GC)/100 mL	0-40	No correlation	(100)
India	Chennai 9.6 million January2021	WWTP, Composite wastewater	UV, Pasteurization, Filtration Corning Spin X-ultrafiltration	Manually (TRIzol for RNA extraction)	2019-nCoV CDC EUA Kit	1.41×104-1.99×104	3983- 5523	Higher viral load than CS	(101)
USA	Virginia 1,700,000 March 2020	Raw wastewater WWTP Grab samples NI	Innova Prep Concentrating method	Nucli SEN Seasy Mag	RT-ddPCR, One-Step RT-ddPCR Advanced Kit, N1, N2, N3	102-105		Trend correlated with clinical data	(101)
USA	Indiana NF November2020	University Sewer Manhole,Raw sewage, Grab	Filtration,Centrifugation	QIAmp Viral Mini Kit	RT-PCR master mix N1, N2	1.0× 104-1.0× 106	0-120	Early detected than clinical	(102)
USA	Las Vegas 1,060,000 March 2020	WWTP Grab samples	Raw influent wastewater and primary effluent composite and grab sample	RNeasy Mini Kit	i Script™ Select Kit, E,N1,N2,ORF1a	104-106	20 and 200	Correlation with clinical data	(103)
USA	Charlotte >2550 November,2020	University plumbing cleanouts & manhole, composite Raw	Electronegative filtration	QIAamp viral mini kit	iTaq™ UniversalProbesOne-Step Reaction Mix N1,	394-2,990,271	400-13,00	Asymptomatic patients detection	(104)
Brazil	Belo Horizonte ∼2,000,000 August 2020	WWTP Influent Composite	Adsorption	RT-qPCR PrepPowerViral DNA/RNA,	iTaq™ Universal probes One Step reaction mix N1	5.6× 101 -2.1×105	0-1200	A similar trend with hospital cases affected by COVID-19	(105)
Sweden	Gothenburg 755,940 July 2020	WWTP Influent, Composite & grab	Adsorption, Filtration	DNeasy Blood and Tissue kit	qPCR Reaction Mix (Invitrogen)	6.7×103 -1.8× 106	0-90)	WS trend peaked when hospitalized COVID-19 increased	(106)
Qatar	Doha 2,503,457 August 2020	Influent WWTP Composite	PEG	RT-qPCRSARS-CoV-2(2019-nCoV)kit N1,N2	Quick-RNA Viral Kits (Zymo Research, Irvine CA, USA Cat. No. R1041)	7.8×103 -5.4× 105	500-2500	Higher than CS	(107)
Brazil	Florianopolis ∼5000 March 2020	Raw sewage	Adsorption	QIAamp Viral RNA Mini kit	One-Step qPCR Quantinovakit, Seegene Allplex™2019-nCoV N1,S,RdRp	Avg 3.1× 105-4.8× 106	NR	WS detected prior to CDT	(108)
Italy	Milan, Turin Bologna 4,998,600 Feb,2020	WWTP Composite raw sewage	Ultracentrifugation	Nucli SEN Smini MAG	Super Fi Green PCR Master Mix RdRp, ORF1ab	2.9× 102 -5.6× 104	3095 in Latium Region and 2186 in the province of Rome	WS dtected before first case by CDT	(109)
Spain	Murcia 716,388 April 2020	Influent, WWTPsComposite	Aluminum hydroxide adsorption– precipitation method with 3% beef extract	Nucleo Spin RNA virus kit	TaqMan one-step master mix (N1, N2, and E)	1×105 to 3.4 × 105	12-622	Detected RNA in low prevalence area	(110)
USA	Virginia, 143,000 September 2020	WWTP, Hospitals,Dormitoriecomposite, and Grab Influent wastewater	Filtration, PEG	QiaAmp viral RNA mini kit and NucleoSpin RNA Plus kit	NF N1, N2, RP	Ct value(30.6-41.9)	NR	Consistency with clinical data	(111)
Japan	Ishikawa and Toyama 465,243 April 2020	Influent wastewater WWTP grab	PEG and NaCl	QIAamp Viral RNA Mini Kit	Prime Script™ N2,N3	1×100 - 3.5 × 104	5-30	Higher than clinical data	(112)
Japan	Yamanashi Prefecture NF May 2020	WWTP Grab	Adsorption-elution, Electronegative filtration	RNeasy Power Water Kit	Probe qPCR Mix with UNG N1, N2	qPCR Mix with UNG (Takara Bio, Kusatsu, Japan)	(1.4 × 102–2.5 × 103 copies/L)	Similar to clinical data	(113)
UAE	June,2020	Raw sewage WWT Composite	Ultrafiltration columns, PEG/TRIzol	ABIOpure Viral DNA/RNA Extraction kits	GENESIG COVID-19 kits RdRP	7.5×102 - 3.4 × 104	0-800	Decrease of WS load related to CS decline	(114)
France	Montpellier≈ 470,000 July 2020	WWTP raw composite	. Filtration, Centrifugation	NucleoSpin RNA Virus kit (Macherey-Nagel)	Primer, Probe based detection N1, N3	100-10,000/100ml	0-75	WS detected SARS-CoV-2 genes before CS	(115)
Spain	Barcelona 2.7 million July 2020	WWTP Composite and grab raw samples	Polyethylene glycol-6000	NucliSENSminiMAG extraction system	NF RdRp,IP2, IP4,E,N1	103-105	2000-8000	WS Detected prior to CDT	(116)
USA	Boston(Massachusetts) NF July 2020	WTPcomposite and Grab samples	PEG+NaCL	TRIzol-chloroform	PCR is used by New England Biolab Master Mix	0Log10-3Log10	0Log10-1Log10	SARS-CoV-2 WS titer higher than CS	(117)

Padilla-Reyes et al. (2022a) found that the concentration of genomic copies of SARS-CoV-2 viral biomarkers (103 and 106 gc/L) were compatible with the reported clinical cases of COVID-19 in three out of four wastewater treatment plants (WTP). The study also revealed that WBEM was capable of giving a signal 2–7 days in advance as early warning, which might be helpful in low-income countries. Another study performed in Mexico found an increasing number of SARS-CoV-2 viral genes in WS two weeks before the clinical cases were raised (82). According to Hillary et al. (2021a), Giraud-Billoud et al. (2021) and Peccia et al. (2020), wastewater viral RNA detection precedes clinical reports by two to five days, three to six days, and six to eight days respectively (65,84,85,88). In another study, Zhang et al. (2020) (86) claimed that the SARS-CoV-2 viral concentration in wastewater was well correlated with COVID-19 clinical cases when samples were collected on day-to-day basis for monitoring the pandemic. According to Nemudryi et al. (2020), the SARS-CoV-2 viral RNA concentration in wastewater samples correlated with dates from sample collection to RT-PCR detection, whereviral genesare detectable in the wastewater samples 5–8 days after collection (68). Zhang et al. (2020) claimed that SARS-CoV-2 in the stool specimen was found significantly elevated than in the serum/blood specimenor nasal swab samples (86).

A recent WBE study conducted over 40 US cities found that a weekly incidence might not be sufficient to support the interpretation of viral concentration in the wastewater (88). Wu et al. (2021) recommended that at least two wastewater samples in a week are necessary to keep the accuracy in COVID-19 trend analysis (88). In another study, Petala et al. (2022) suggested that a weekly-based sampling method for viral quantification with fixed sampling times could be scheduled to reduce the day-to-day deviation (89). In addition, they strongly proposed that wastewater sample test results should be validated with clinical data.

4. Conclusion

WBEM has the potential to detect hotspots, identify the prevalence, and predict early warning. On the other hand, CDT can be used to isolate positive patients, mass vaccination, and quarantine measures to limit direct, indirect, or close contact. In the context of making the CDT method more cost-effective and efficient, it is important to improve in terms of rapidness, sensitivity, and portability of the analyses to demonstrate a functional diagnostic tool for detecting cases of positivity. It is also noteworthy that, the presence of SARS-CoV-2 in the community will be detected earlier by the WBEM than by the CDT. Hence, the dual monitoring of COVID-19 by using WBEM and CDT will immensely help control the spread and threat of the COVID-19 global pandemic.

5. Ethical Statement

No ethical statement is required for this review article.

6. Funding sources

This study was supported by the Water Aid Bangladesh, North South University, International Training Network of Bangladesh University of Engineering and Technology (ITN-BUET) - Centre, Noakhali Science and Technology University. PB and MTI would gratefully acknowledge the Life Science Technology Platform, Science for Life Laboratory for the seed funding to initiate the wastewater-based epidemiological studies for SARS-CoV-2 in Bangladesh.

Uncited reference

28., 34., 40∗∗., 45., 81., 87..

Declaration of interests

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.

Appendix A. Supplementary data

The following is the Supplementary data to this article: