Importance of wastewater-based epidemiology for detecting and monitoring SARS-CoV-2

Abstract

Coronavirus disease caused by the SARS-CoV-2 virus has emerged as a global challenge in terms of health and disease monitoring. COVID-19 infection is mainly spread through the SARS-CoV-2 infection leading to the development of mild to severe clinical manifestations. The virus binds to its cognate receptor ACE2 which is widely expressed among different tissues in the body. Notably, SARS-CoV-2 shedding in the fecal samples has been reported through the screening of sewage water across various countries. Wastewater screening for the presence of SARS-CoV-2 provides an alternative method to monitor infection threat, variant identification, and clinical evaluation to restrict the virus progression. Multiple cohort studies have reported the application of wastewater treatment approaches and epidemiological significance in terms of virus monitoring. Thus, the manuscript outlines consolidated and systematic information regarding the application of wastewater-based epidemiology in terms of monitoring and managing a viral disease outbreak like COVID-19.

1. Introduction

COVID-19 has emerged as a serious infectious disease emerged as a result ofSARS-CoV-2 virus (severe acute respiratory syndrome coronavirus 2) outbreak. COVID-19 infection was observed for the first time in Wuhan, the capital of Hubei Province, China [1]. Subsequently, the virus transmission led to a global outbreak casuing a pandemic situation as declared in March 2020 by the World Health Organization (WHO) [2,3]. The SARS-CoV-2 is mainly enveloped and harbors a positive-sense single-stranded RNA with agenome similarity of 82% on comparing with SARS-CoV- 1 [4]. COVID-19 covers a range of infection including asymptomatic, mild and/or severe pneumonia that might also cause severe organ malfunction [5,6]. The binding receptor for SARS-CoV-2 is ACE2 followed by receptor-mediated endocytosis leading the development of the infection [7]. Host receptor ACE2 is expressed in the gastrointestinal lining allowing the phenomenon of viral replication [8]. On clinical evaluation, the SARS-CoV-2 expression was reported in the gastrointestinal lining [[9], [10], [11]]. Clinical stool samples of GI infected patients were tested positive for SARS-CoV-2. Notably, the screening-detected significant positive cases in most of the patient samples with significant RNA copies in the late phase of the COVID-19 infection [[12], [13], [14]]. Mostly common source considering the pool screening of SARS-CoV-2 RNA is wastewater. Wastewater is a form of polluted water comprising the man-made generated waste particles and rainwater runoff leading to the formation of sewage. The classification of wastewater is based on the form of waste generation, specific source, domestic and industrial sewage. On separating the inactivated virus from the wastewater, low level of persistence might also lead to recurrence based on their survivability in the wastewater facilities, stormwater and contamination of drinking water [105]. Wastewater contains a wide range of contaminants/pollutants that are characterized on the basis of physical, chemical, and biological particles. Moreover, the concentrations and physicochemical characteristics of these substances are dependent on the source of origin. Domestic sewage is a heterogeneous mixture comprising water saturated with organic and inorganic entities and diverse pathogenic microbes (US EPA 2003). Moreover, the pathogenic microorganisms also include different strains of bacteria, viruses and parasites that can lead to the development of numerous diseases (see Fig. 1 ).

Fig. 1.

Schematic representation of workflow for screening of SARS-CoV-2 in waste water samples.

Wastewater-based epidemiology (WBE) an emerging approach has been under consideration for regulatory monitoring of pollutants and specific biomarkers accumulated in wastewater followed by the qualitative and quantitative analyses of the inhabitant's activity in a particular wastewater catchment. WBE is useful in determining the substance usage and exposure in case of environmental pollutants and estimated prevalence of a range of diseases based on the wastewater constituents including biomarkers and environmental pollutants [15]. Several studies have reported the persistence of SARS-CoV-2 in the clinical samples for a specific period of COVID-19 in the wastewater, including countries like USA, Australia, France, Spain, Italy, India and China [16,17,[19], [20], [21],118]. WBE application to screen the prevalence of SARS-CoV-2 has been investigated for dynamic monitoring and preventing community based outbreak of the virus infection. Earlier, WBE application has shown positive detection to screen the virus associated pathogens [22,23], including the WHO acute flaccid paralysis program (https://apps.who.int/iris/handle/10665/67854; https://www.who.int/wer/2015/wer9021/en/) [24]. The presence of SARS-CoV-2 RNA in wastewater with the support of multiple studies has highlighted the significance of viral circulation throughout the pandemic period, including the monitoring of pre-and post-lockdown phase. Mostly, the qualitative analysis reported the prevalence of the virus based on the site-specific incidences, proceeding from 7 to 21 days with a major outbreak of the new COVID-19 infection in the population. But, the quantitative evaluation for screening the virus was mainly dependent on clinical studies. Hence, clinical investigations were performed with the screening of fecal matter based on infection outbreaks and specific population sites. Incidence of gastrointestinal symptoms was reported in two independent cohort studies with 19.4% showing diarrhea-like symptoms in the initial phase [25]. Further, a cohort study confirmed half of the positively infected patients shed the virus in the fecal matter followed by wastewater screening [26]. Moreover, the virus was detected in both asymptomatic and symptomatic people, and further evaluation assessed the presence of the SARS-CoV-2 RNA copy in the feces compared to the negative respiratory swab test reports [27]. The confirmatory presence of virus RNA was reported in 10.1–82.0% patients based on clinical investigation of the fecal swabs [[28], [29], [30], [31]] compared to respiratory swabs which tested negative or undetected for SARS-CoV-2 [30]. Virus quantification of the clinical specimens revealed a high genome copy number (10⁸ copies/swab) along with the high values at the end or late phase of infection [[32], [33], [34]]. The time period for determining the nucleic acid quantitatively varies among cohort, but prolonged RNA with respect to infection monitoring has been highlighted for a time period of 33 days after the onset of the infection manifestation [30,35,36]. On the contrary, some studies found that the quantitative estimation of virus in the fecal sample was not in proportionate with the infection distribution among the population considering the degree of infection [37,38]. Studies have provided the insights of virus RNA copies in the stool based on clinical sampling and testing [39,40], but many remained undetected even when the infection is positive for the individual. The survival period of the virus in the gastrointestinal system depends on facilitative persistence over the respiratory system based on the ACE2 expression in intestinal enterocytes and low viral clearance [41]. Further, the fate of SARS-CoV-2 in the treatment plants is crucial in terms of virus inactivation which includes different stages of water treatment to ensure effective control management and prevent further occurrence of the outbreak [97]. The screening of SARS-CoV-2 survivability in the wastewater, fecal and oral mode of transmission, and estimation of viral-titer will also improve the method of clinical evaluation. Hence, the application of WBE in determining the infection load can improve the health-based pandemic control and upscale preventive measures to restrict the spread of variant-based outbreaks in the community.

1.1. WBE application in SARS-CoV-2 surveillance

WBE monitoring is based on the detection of genome of SARS-CoV-2 especially the virus RNA in the wastewater in determining the virus presence and severity of infection in a particular population [42]. The feasibility of WBE is dependent on wastewater screening which is a composite source of biological samples restricted to an entire community. Several WBE studies have drawn the attention towards the prevalence of SARS-CoV-2 genomic copies in the wastewater in the preliminary phase of the virus infection [18,24,64]. WBE could be especially informative provided that asymptomatic and mild to moderate COVID-19 infections are unlikely to be tested positive in the clinical surveillance. In such case, evaluation of positive cases out of the total sample depends on mathematical calculations with specific models. The models are proved to be efficient in monitoring the dynamics of disease outbreak as well as management. Hence, WBE can be applied to estimate the load of undiagnosed infections at the community level with the help of mathematical models, and also refining the estimated case-fatality rates with respect to infection load in the community.

Based on clinical investigations and technological methods, WBE could play a significant role in regulatory assessment of SARS-CoV-2transmission based on community level monitoring in the initial phase, exponential, and re-emergence phase at epidemic level. This particular approach underscores the earlier measures in environmental monitoring, like poliovirus RNA, to delineate mechanistic models of pathogen transmission [119]. In WBE approach, the prevalence of SARS-CoV-2 infections in a population could be detected by enumerating the SARS-CoV-2 RNA confined to a particular sewage site followed by determination of mass balances based on virus shedding with the help of population and sewage flow rate data sets. Moreover, the specificity still requires to be modified based on the nature of the virus strain and target gene. Thus, the information can further be forwarded to public health responses to restrict or prevent the outbreak.

The current pandemic provides a practical opportunity to field-test the hypothesis of WBE application based testing and infection control to prevent further transmission in the population. Several research groups across the globe have implied the significance of WBE in controlled monitoring of the virus spread. Moreover, quantitative analyses of virus RNA in sewage and human infections are dependent on certain spatial and temporal variables. Significantly, the quantitative analyses should be examined in urban sites with centralized wastewater facilities rural sites and low-income zones with decentralized wastewater infrastructure. Assessment of variables and uncertainty in the diverse wastewater sites require the systematic approach and validation of methodologies across research groups. Moreover, to improve the potential WBE applications, a comprehensive effort to coordinate technological settings and data enumeration can be established to maximize the yield efficiency of WBE for the present and future outbreaks of infectious diseases.

Comparative analyses of WBE assessment is dependent on a standardized approach comprising a robust sample designing, techniques to improve viral recovery and estimation followed by virus RNA detection from the wastewater [64]. To delineate the viral recovery and significantly determine the prevalence of virus RNA in wastewater for WBE, a control for the recovery of virus is important. The control is mainly a non-SARS-CoV-2 virus introduced in a sample of wastewater with predetermined concentration before the screening of the sample. Further, evaluating the result of the virus recovery with respect to SARS-CoV-2 presence was performed with the technical support and laboratory procedures across different laboratories. The Water Research Foundation (WRF) further supported a cohort study comprising 32 different laboratories in the U.S. including a set of 36 individual standard operating procedures [59]. But, the recovery analyses prevailed insignificant on comparing due to the inclusion of different set of preparation procedures. Generally, multiple WBE preparation for SARS-CoV-2 analyses have not included control recovery group and data information related to the recovery based experiments. Kantor et al., 2021 further proposed the obstacles associated with the quantification of SARS-CoV-2 RNA recovery from the collected sample and highlighted the requirements for clinical investigations [58].

1.2. Survivability of SARS-CoV-2 and its transmission through wastewater

Individuals infected with SARS-CoV-2 progressively because the shedding of the virus during the recovery and/or post recovery period which is associated with the virus RNA concentrations in wastewater. The RNA concentration can be assessed as an indicator to determine the infection load particularly for monitoring purpose. However, the quantitative assessment of evaluating a community's viral load and the advantage to public-health policy and local governance decisions is strongly inclined towards constant sewer shed RNA level monitoring. On the basis of reduced RNA level concentrations, wastewater-based epidemiology can once again be applied as an indicator of a resurgence of the viral transmission. The SARS-CoV-2 virus shedding through fecal matter is known, but it is in still not known regarding the nature of the shedded virus i.e chances of infectious state and the period of retaining the infectious state in the wastewater. Reports have suggested that an individual infected with SARS-CoV-2 can shed more than million copies of viral genome per litre of the wastewater [16]. The virus is enveloped, quite unstable in water and susceptible to inactivation. Viability analyses have highlighted the presence of SARS-CoV-2 in the fecal sample for 2–6 hrs at room temperature and more than 48 hrs in the fecal matter of children [42]. Further, research studies have estimated the survival of SARS-CoV-2 i.e. survival up to 3–4 hrs in the urine sample was reported in COVID-19 infected individual [43]. The COVID-19 infection based on shedding of the virus along with water contamination implies the uncertain route of fecal-oral transmission of the virus [44]. But, estimation of infection requires specific mathematical models to delineate the trend in infections and proper monitoring of the outbreak. Krivoňáková et al., 2021 discussed the application of regression models like simple linear, double squared root, and square root-Y logarithmic-X to study the association of wastewater data with the data of SARS-CoV-2 infection [110]. Similarly, Ahmed et al., 2020a mentioned a mathematical approach to estimate the rate of infection based on wastewater screening [17]. The estimated number of viral RNA copies/g of feces was reported to be 10⁷ [45,111]. Another approach was applied based on RNA copy number per litre of wastewater with respect to RNA copy numbers released per person to the sewage water [[112], [113]].Wastewater samples were tested for the presence of SARS-CoV-2 on different time periods by targeting the viral E and RdRp gene with the application of RT-qPCR which reported a high concentration of viral load was estimated in the study [18]. Survival quantification for the water site or zone of water-linked virus accumulation estimated 1.6–2.1 days at a standard temperature [45]. Notably, the viability of the virus in wastewater has been explored under the conditions of low temperature [46,47]. The virus RNA concentration based on different categories of water background determines the estimation of copy number in association with the trend in infection rates including the variants of concern. The viability depends on several factors including temperature, diverse pool of microorganisms, and organic matter in the wastewater. On evaluation, it was reported that the sludge from the wastewater can be primarily monitored for screening of the virus. For quantification the concentration of the virus was measured under different wastewater treatment conditions. Specifically, a high copy number was observed in both types of sludge including primary and thickened compared to the influent wastewater [48]. The survival of the virus subjected to different wastewater treatment methods can also act as a source of contamination and transmission depending upon the efficiency of the treatment approach. Hence, the virus load in the wastewater matrices should also be considered prior to and after the treatment to assess the specific survivability to determine the threat of virus infection in a particular zone or area. The World Health Organization has highlighted the importance of tap water treatment in virus inactivation, emphasizing the susceptibility of pathogens towards chlorination and ultraviolet based disinfection. Similar methods with improved filtering capacity can be applied to restrict the transmission of corona viruses through treatment plants. Multiple reports that have suggested the importance of different stages of waste water treatment approaches to limit the viral load compared to the initial concentrations. These approaches mainly includes anaerobic system, moving bed biofilm reactor, sequencing batch system, secondary treatment along with tertiary disinfection methods using peracetic acid, high-intensity UV lamps, or chlorine [89].These treatment methods reduced the concentration of viral copies from high copy load to an estimated level of 102–105 copies per litre. Moreover, chlorine based disinfection method is also recommended for effective elimination of the virus comprising the aqueous phase of the influent; however, the stool particles consisting the SARS-CoV-2 virus were considerably not susceptible to chlorine disinfection compared to aqueous phase [[49], [50], [51], [52], [53]]. In course of detecting the virus several concentration methods have been applied to target the virus genes and further estimate the virus copy number in the wastewater (Table 1, Table 2 ).

Table 1.

Application of different concentration methods to screen the SARS-CoV-2 virus in wastewater.

Country	Type of Wastewater	Virus Concentration Method	Virus Target Gene	Copy Number/Concentration per Litre	References
India	Untreated Wastewater Untreated Wastewater	PEG Precipitation PEG Precipitation	ORF1 ab, N, S ORF1 ab, N, S, E, RdRP	8.05 X 102 –	[88,89]
China	Untreated and Treated Wastewater	PEG precipitation	ORF1, N	–	[85]
USA	Untreated Wastewater Untreated Wastewater	PEG precipitation (PEG 8000) Ultrafiltration and Adsorption	N1, N2, N3 ORF 1a, S	60-300 copies 4 × 103–7 ×  103	[35,122]
Australia	Untreated Wastewater	Ultrafiltration	N	2 × 101–1.4 ×  102	[86]
France	Untreated and Treated Wastewater	Ultracentrifugation	RdRP, E	5 X 104	[18]
Germany	Untreated Wastewater	Electronegative membrane filter	ORF1 ab, N, S	3.9 X 1011–1.2 X 1015	[123]
Spain	Untreated Wastewater Untreated Wastewater	Aluminum hydroxide adsorption-precipitation Aluminium flocculation and precipitation	N N	1.4 × 105–3.5 × 105 104–105	[80,124]
Italy	Untreated Wastewater Untreated Wastewater	PEG precipitation Ultrafiltration	ORF 1 ab ORF ab, N	– –	[19,125]
Israel	Untreated Wastewater	PEG and Alum precipitation	E	–	[87]
Iran	Untreated Wastewater	PEG precipitation (PEG 6000)	ORF 1 ab, N	–	[126]

Table 2.

Clinical evidence of SARS-CoV-2 screened from wastewater across different countries from the year 2020–2021.

Country	Month/Year	Sample	Analysis	References
India	June/2020	Wastewater from hospital	•SARS-CoV-2 RNA detection was 100% in wastewater •Virus load 10 fold higher in wastewater, effluent and sewage •Early RNA detection preceding the increase in infection rate from 9 to 12 days	[88,90,91]
Brazil	May/2020	Wastewater	•SARS-CoV-2 RNA concentration was 41.7%–100% in wastewater •Virus screening was conducted with samples based on infection rate and detectable possibilities •Screening was in coherence with positive reports and epidemiological data	[[72], [73], [74],127]
USA	March/2020	Urban treatment plant, wastewater from catchment points	•High positive reports of SARS-CoV-2 infection •Viral RNA concentration was 40% in the screened samples •High virus copy number preceded infection rates in 1–12 days	[35,[75], [76], [77], [78]]
Netherlands	March/2020	Wastewater	•Viral RNA was detected in wastewater from six major cities •Virus detection rate was found to be 69% and detectable count preceded the infection in 4–8 days •Significant expression was observed in early detection	[79,128]
France	July/2020	Wastewater treatment plant	•Viral RNA was detected in the wastewater (100%) •The detection of genome copy was fifty in the post-lockdown phase in the month of June •Early detection was reported based on clinical data which preceded 2–5 days of COVID-19 infection •High copy number was reported in the wastewater in accordance with the increase COVID-19 cases	[80,100]
Germany	April/2020	Wastewater treatment plant	•High expression of viral RNA in the sample (100%) •Sequencing reported high viral copy number with the surge in infection cases •Total load of virus copy number was in coherence with the clinical data of COVID-19 infection	[81]
Italy	April/2020	Wastewater	•Viral RNA was detected in the sample water (50%) positive cases •Viral RNA concentration was detected significantly in early clinical evaluations	[82,83]
China	July/2020	Septic tanks	•SARS-CoV-2 RNA was detected in 60% out of the total sample pool •Viral RNA was not observed significantly in effluent after disinfection	[85]
Bangladesh	August/2020	Wastewater	•High load of viral RNA was reported	[109]
Australia	April/2020	Wastewater	•Viral RNA concentration was 22.2% in the wastewater	[86]
Israel	Not reported	Wastewater	•Viral RNA concentration was 38.4% in hospital wastewater	[87]

The approved guidelines for the disinfection of wastewater based on WHO or China Centers for Disease Control and Prevention recommendations may require to be improvised. According to the relative vulnerability of the virus, present wastewater procedures may require to improve in order to reduce the inefficiency in reducing the virus concentration to a protected virus copy numbers. Even after the application of wastewater treatment procedures, the remaining population of 1.8 billion still susceptible to the fecal-contaminants as a source for infection. For example, wastewater disinfection in India is only half compared to the volume of wastewater generated per day. In order to screen the wastewater for SARS-CoV-2 a standard method like RT-qPCR was practiced for detection and monitoring. The RT-qPCR based detection was mainly based on targeting the protein encoding genes including E, N, S, and also ORF1ab/RdRp [106].The cycle threshold (Ct) for targeted genes was less than 31 for most of the positive cases corresponding to the test of RT-qPCR [107]. Further, quarantining infected individuals may not reduce the mitigate risk based on improper disinfection of the wastewater [54]. Hence, clinical data related to WBE and its association with SARS-CoV- 2 has unfolded the significance of infection susceptibility and transmission through wastewater.

Comparatively, wastewater treatment has been mostly regulated with respect to the reduction of enteric viruses (non-enveloped) which also spreads through fecal-contamination, but stringent rule lack regarding enveloped virus treatments. However, non-enveloped viruses are associated with high risk compared to viruses like SARS-CoV- 2, which comprise an envelope. Wigginton et al., (2015) justified, “Based on the cumulative survivability data, it is plausible that an enveloped virus excreted in human feces or urine could survive in aqueous environments for periods of time that is relevant to the wastewater and prior to its treatment”. Higher levels of infective viruses would be expected in a wastewater treatment plant influent when there is a large incidence rate in the community and when wastewater temperatures are cooler” [55].

The susceptibility of SARS-CoV-2 infection based on the commercial application is also related to the persistence of the virus in the wastewater during the treatment process until the phase of water consumption by the humans. The detection of SARS-CoV-2 in feces depends on the duration of survival including the exposure period of the temperature and treatment conditions. Notably, the survivability of the virus is significantly higher until the wastewater treatment. Although the efficiency of wastewater treatment in reducing the virus concentration lack prominent evidences, clinical reports published indicate that the present treatment method reduces the viral load below the detectable concentrations. The discharge of treated wastewater into the open spaces or surface waters followed by the participation of human activities i.e drinking water usage improves the survival of the virus irrespective of the treatment process.

The risk of infection transmission before the arrival of the wastewater in the sewer lines lies in different sites including residential places, hospital areas, as well as the healthcare settings based on the interconnected pipelines of the plumbing system. Even aerosols harboring the virus can pass the building sites, without any barrier [56]. The contamination of surface runoffs or consumable water with SARS-CoV-2 can also be assessed for quantification and in application to indicate the risk of virus spread in the particular zone. Hence, the significance of wastewater with respect to SARS-CoV-2 transmission and effective disinfection approaches through wastewater treatment requires clinical studies for the recommendation of WBE for the monitoring purpose of COVID-19 infection.

1.3. WBE application in screening of SARS-CoV-2

Due to the limitation of well defined approaches, WBE linked SARS-CoV-2 monitoring is comprehensive and requires clinical and technological expertise [[57], [58], [59]]. The WBE approach requires wastewater sampling, extraction of viral RNA, viral load determination followed by data analysis. Generally, untreated wastewater and primary sludge are two sources for sampling and testing of wastewater. Investigations reported a higher virus RNA concentration by the two-three folds on the clinical sampling of primary sludge compared to untreated wastewater [57,60,61]. Hence, primary sludge can be screened for concentrating and detecting the virus based on reduced volume and low inhibitory factors which might hinder the concentration determination requirement for sampling purposes. The frequency of sampling further requires the estimated range to assess the virus detection through WBE approach. Moreover, screening the samples once or twice in a week for the screening of the virus SARS-CoV-2 in wastewater may be sufficient, while the dynamics in infection rate could indicate early warning of the infection. The dynamics in infection rate including the factors like virus RNA stability, persistence, the effect of temperature, and relative proportion after the lockdown was clinically evaluated [[98], [99]]. A study based on the application of wastewater screening highlighted change in virus concentration in association with the dynamics of epidemic stages [57,62].

According to the guidelines of Centers for Disease Control and Prevention (CDC) the clinical sample of the virus should be maintained at 4 °C and further the viral assays should be performed within 24hours. The viral aliquots should be maintained at −70 °C and limit the frequent cycles of freeze thaw. Few reports demonstrated the stability of virus RNA at 4 °C for a period of 14 days [63]. A study reported the decay of virus RNA through linear decay graph study at 4 °C within a period of 28 days in simulated wastewater samples comprising the virus from the nasopharyngeal swab of an infected patient [120]. Hence, it is highly recommended to process the sample in a period of 48–72 hrs to prevent the degradation viral RNA in wastewater [64].

Wastewater consists of a heterogeneous mixture of chemical and biological compounds, which not only reduces the viral recovery but also leads to poor reproducibility leading to the hindrance of efficient screening and quantification of virus RNA from the wastewater samples. External matters and dissolved constituents that forms the concentrated part of the sludge through water way reduces the efficiency of downstream process of viral RNA extraction and detection. The concentration procedures should be simple, rapid, effective and inexpensive and should be able to filter out enormous amounts of wastewater. For example, precipitation through polyethylene glycol, aqueous two-phase partitioning (PEG-Dextran system), application of electronegative membranes (EM), ultrafiltration, and ultracentrifugation are all common concentration procedures for SARS-CoV-2 by WBE. Clinical evaluations were performed with the help of ultracentrifugation by targeting the inactivated SARS-CoV-2 virus. For example, the Amicon Ultra-15 method was applied to screen inactivated SARS-CoV-2 virus [104], murine hepatitis virus [101], and bovine coronavirus [102]. Similarly, Centricon Plus-70 was applied to screen inactivated SARS-CoV-2 [104], F- specific RNA phage [79], and bovine coronavirus [103]. Further, precipitation and membrane adsorption with AlCl3 and MgCl2 was performed to recover the inactivated SARS-CoV-2 virus [104]. Also, PEGylated precipitation was performed to recover the inactivated SARS-CoV-2 virus, bovine coronavirus, gamma-irradiated SARS-CoV-2 virus [104].

1.4. SARS-CoV-2 RNA extraction from wastewater

The RNA extraction from the SARS-CoV-2 virus involves major steps including envelope disintegration followed by extraction and purification of RNA from DNA and proteins [65]. The envelope disintegration can be achieved through different approaches like mechanical, chemical, or enzymatic methods [[65], [66], [67]]. Further, the stability of the genome also depends on the pH, ionic strength, and RNA degrading enzymes like RNAses. Hence, the purification step is crucial with respect to RNA integrity and preventing RNA degradation. Notably, the phenol-chloroform method and/or solid-phase extraction process are significantly implemented for virus RNA purification from the samples of wastewater.

1.5. Application of PCR method to screen SARS-CoV-2 RNA and proteins

Screening of the virus RNA can be achieved with the application of exponential amplification studies. Zheng et al., (2020) proposed the application of molecular biology approaches like PCR specifically, RT-qPCR for the detection of SARS-CoV-2 [68]. TheRT-qPCR method mainly targets the structural protein encoding genes like envelope, nucleocapsid, spike and RNA-dependent RNA polymerase along with other regions, for example open reading frame 1ab (ORFab). A protocol has been recommended to screen the SARS-CoV-2 virus based on the recommendations of WHO (https://www.who.int/docs/default-source/coronaviruse/whoinhouseassays.pdf). Notably, the E and N genes are mainly targeted for the virus detection based on their conserved sequence in many corona viruses. Nevertheless, the S and RdRp genes are specific to the virus. Hence, both or single gene screening may be significant for the detection of the SARS-CoV-2 RNA from the rest of the corona viruses [69].

Further, screening of different genes may also improve the viral assay specificity and efficiency with a reduction in the errors for the identification of uncharacterized virus genome or the virus. Subsequently, with the rise of new SARS-CoV-2 variants comprising alpha, beta, gamma, and delta, it is important for wide application RT-qPCR method based on its property of quality detection and to differentiate different SARS-CoV-2 variants at the scale of community surveillance. But, still significant improvements are required in RT-qPCR assays in terms of sensitivity and specificity in differentiating the predominating variants compared to other viruses in the wastewater. An alternative to RT-qPCR research groups have demonstrated the sensitivity of droplet digital PCR for SARS-CoV-2 screening. A cohort study with digital PCR reported low assay limit of detection compared to RT-qPCR [108]. In addition, the performance of ddPCR was not affected by the potential PCR inhibitors present in the wastewater sample, and demonstrated higher analytical output with limited false reports compared to RT-qPCR [94]. Heijnen et al., 2021 has shown the efficiency of ddPCR in SARS-CoV-2 detection based on a single amino acid substitution. The mutation resulted in asparagine to tyrosine substitution at 501 position resulted in higher binding affinity of SARS-CoV-2 towards ACE2 receptor, and the variant further came to known as B.1.351 [95]. The study included a differential detection of the B.1.351 variant from the wild type, and finally, ddPCR efficiently detected the low concentration (0.5%) of B.1.351 variant from the heterogenous mixture of the wastewater sample [95]. Similar test was performed to detect the B.1.1.7 variant proportion in the wastewater collected from cities like Amsterdam and Utrecht. Further, ddPCR was applied to detect different variants of SARS-CoV-2, and to the analysis it was proved to be better and efficient compared to the RT-qPCR based SARS-CoV-2 screening. Subsequently, a comparative study between ddPCR and RT-qPCR with the SARS-CoV-2 positive patients was performed after taking the swabs from the rhino-pharyngeal site. The sample dilution for RT-qPCR was 1:10 and sequenced against CDC-validated SARS-CoV_N1 primers. Comparatively, samples for ddPCR were 1000 fold diluted which was higher than RT-qPCR. Subsequently, ddPCR was found to be highly sensitive and robust than RT-qPCR. Notably, the linear regression study highlighted the less susceptibility of ddPCR (r² = 0.9420) towards inhibitory factors of PCR compared to RT-qPCR (r² = 0.0919) [96]. The ddPCR reported positive for SARS-CoV-2 in the undiluted as well as the samples with tenfold dilutions, which was not observed in RT-qPCR [96]. Hence, the accuracy and efficiency of ddPCR based approaches can be applied to quantify the SARS-CoV2 rapidly.

1.6. Interpretation

In clinical studies, the rate of viral shedding and associated parameters are significant because any kind of change in a variable factor might provide error-prone data-based sampling, specific stages of infection, rate of transmission, viral load, calibration of virus genome copy numbers, infection threshold and difference in a particular or combined community. The calibration of genome copies of SARS-CoV-2 per litre in the sample wastewater, the significant proportion of affected individuals or the prevalence of the infection can be estimated. Further, calibration of genome copies of SARS-CoV-2 per litre in the sample wastewater including variable factors should be considered for screening and clinical evaluation of the infection. But, the calibration curve determination for viral studies is should be independent of the recovery rate. Moreover, the percentage of viral infection in a certain population depends on the estimated viral loads, rate of transmission, and the significant RNA concentration in the fecal matter of the affected population as well as the measured regular generation or collected pool of stool per capita.

Once the virus genome is quantified with the application of RT-qPCR, specific Ct value can be further assessed for estimating the virus titer per unit of sample wastewater or even samples taken from sludge output based against a specific standardized curve. The template volume of the template used for amplification is calculated considering the conversion factor and also the RNA recovery. The quantification of the RNA copy number per unit of wastewater sample was mainly determined by RT-qPCR. The virus concentration estimated by comparing the Ct value of the pool sample against a standardized curve with respect to a specific assay on comparing with a positive control provided the specific dilution required for amplification. Further, a standardized curve is prepared by using a control with six times dilution for the determination of minimum detection limits (MDL) per unit amplification reaction followed by the evaluation of the efficiency of the reaction.

1.7. Protein based SARS-CoV-2 screening

The SARS-CoV-2 virus screening can also be performed by targeting specific proteins based on the concentration and stability of the proteins [70]. Mainly, the structural proteins along with other nonstructural proteins can act as a plausible target for viral detection. The nucleoprotein can be considered for clinical testing, but it is also limited due to the common nature of the gene throughout the corona virus family. The alternative target can be the S protein based on its sequence divergence among the viruses of the family. Specifically, the S protein is important for the virus to enter the host via the ACE2 receptors. Hence, it facilitates the virus infection and acts as clinical target for vaccine development [121]. Although protein-based detection is limited because of the change in the nucleic acid sequence which can be advanced through primers or targeted regions, but not similar to antibodies for protein detection.

Application of affinity ligands bearing high specificity and sensitivity can be implied for screening, like, ELISA based detection method. Rapid antigen based method to detect the virus in the clinical samples. Moreover, antibodies were tested against the viral nucleoprotein through Western Blots in order to create a multiplex paired-antibody amplified detection (MPAD) assay for detecting the virus [71]. But, still more evidences are required to design the target specific ligands with optimum specificity and affinity for screening the persistence of SARS-CoV-2 in the wastewater.

1.8. International surveillance on WBE based screening of SARS-CoV-2

Virus shedding and screening through WBE method has been practiced on different time periods of the pandemic waves as well as lockdown episodes. The genome copy numbers were detected in the catchment points comprising drain outlets and sewage water. Notably, the majority of the countries initiated the monitoring of WBE in the first quarter of 2020. A first clinical surveillance study showed the prevalence of the virus RNA in the wastewater sample upon investigation in Niteroi municipality of Rio de Janeiro, Brazil. The screening of wastewater reported a viral load of 41.7% in the sample pool which later showed higher prevalence with 100% RNA load during the period of April to May 2020 [72]. Similarly, positive cases were observed in the wastewater collected from six major six cities of the Netherlands, targeting three nucleo capsid genes followed by the gene encoding envelope protein. The RT-qPCR reported significant expression of targeted genes in the sample validating the presence of positive cases in the particular zonal region [79]. In Australia, viral RNA quantification revealed a positive rate of 22.2% in the wastewater collected from Queensland in the month of March and April [86]. The wastewater samples collected from the urban treatment plant of Massachusetts were screened to detect the virus RNA by targeting the N gene with the help of RT-qPCR followed by the S gene validation. Significant expression of the viral RNA was reported during the period of March 2020 validating the sequential event of higher positive cases clinically reported in Massachusetts [75]. In the USA, the WBE was conducted within a population of 2 million confined to some selected cities. Further, high gene expression was reported in the wastewater samples collected from the outlets of water treatment sites, influent catchments, and interceptor points. The RT-qPCR results revealed a 40% positive case of virus genome which showed significant association with the high incidence of virus infection cases including Syracuse and other locations like Onondaga County in May 2020 [76].Similarly, primary sludge in the urban treatment plant of New Haven, Connecticut was tested for the viral load. The virus RNA concentration per unit volume was reported to be 1.7 to 4.6 x 10³where approximately 96.5% of all CT values were less than 38 for the primer sets against specific nucleoprotein as well as both replicates [77]. Further, WBE was also performed within a range of 74 days in the City of Bozeman, Montana. The virus RNA concentration was tested based on the onset of positive cases and post incidence of infection cases. A clinically relevant surge was not reported in the period of April to May 2020, but a specific surveillance system based on RT-qPCR was reported in the study [78].

WBE method of surveillance for SARS-CoV-2 RNA was also conducted in different parts of the European continent. In France, significantly higher expression of virus genome was found in the wastewater collected from the treatment sites of Paris and Montpellier. Notably, a high copy number of RNA was reported in the study, but a significant correlation was not present including the period of the pandemic surge and post-lockdown [100].

Similar application of WBE in Germany demonstrated the trend of high RNA concentration by screening the wastewater collected from treatment plants of nine different cities during the onset of first wave of COVID-19. Based on sequencing and statistical analyses a strong correlation was reported between high virus genome copies and incidences of COVID-19 infection from specific collection catchment points [81]. Further, half of the clinical sample collected from different parts of Milan and Rome presented higher number of virus RNA copies [82]. Also, molecular epidemiological studies validated the prevalence of the virus by screening the wastewater for the genome expression collected from Milan, Turin, and Bologna in the early phase of the pandemic [83]. WBE studies conducted in China showed varied survivability of SARS-CoV-2. A surge in copy number of RNA was reported in aerosols and wastewaters near hospital zone restricted for treating COVID-19 patients. The surge in RNA was a result of viral spillover from the hospital environment in the form of respiratory droplets and significant patient load near the water sites of the hospital [84]. Also, elevated virus load of (0.5–18.7) × 10³ copies/L was reported in the septic tanks of Wuchang Cabin Hospital even after disinfection [85]. The Noakhali district in Bangladesh bearing the highest load of infection reported the prevalence of higher virus RNA copy numbers during the period of July to August 2020. A preliminary study with the hospital wastewater from Tel Aviv and Jerusalem reported 38.4% of samples harboring the virus RNA with respect to the infection rates and patient hospitalization [70].

1.9. National surveillance on waste-water based epidemiology of SARS-CoV-2

WBE based virus screening was conducted with the help of wastewater samples collected from the hospitals in India. Mainly, the study was performed between May to June 2020. Notably, high genome copy number was reported from the samples of untreated wastewater during the initial phase of lockdown. The prevalence of viral RNA copy number was 100% on screening the clinical samples, and significant correlation was observed with the high incidence of the virus infection [88]. In addition, an investigation demonstrated the RNA concentration to be ten-fold higher along with a two-fold increase in the infection rate in the month of May 2020 [89]. WBE method for virus monitoring was successfully conducted from with the samples collected from the fragmented sewerage system in Jaipur. The study reported the presence of genes encoding for envelope, RNA dependent RNA polymerase, nucleocapsid, and ORF1ab gene where the nucleocapsid gene number was higher compared to other genes. Genome concentration in the effluent was determined under different treatment conditions of the wastewater [90]. Further, the newly emerged different strains of the virus marked as variants of concern (VOCs) were also monitored across 11 distinct water treatment plants in Jaipur. Successful validation reported the prevalence of different strains specifically B.1 and B.1.617.2 in the consecutive weeks of wastewater sampling [91]. The clinical validation of the virus genome was further accepted as a monitoring parameter in association with the emergence of COVID-19 cases. Moreover, a large-scale implication of WBE-based virus monitoring still requires significant advancement in India. Thus, an effective method can be practiced with respect to COVID-19 monitoring based on the emergence of new variants, and restricting the recurrence ofCOVID-19 infections considering the geographical and diverse population groups.

1.10. Opportunities for further research

Application of WBE can be an alternative approach in terms of screening and advancing the detection of SARS-CoV-2 based on community level and geographical based monitoring and prevention. On considering the limitations, still WBE needs technical modifications for further application in terms of pandemic surveillance. The importance of WBE has been demonstrated through retrospective studies for early warning of the infection, but real time application still requires frequent sampling, effective collection of samples, technical application, and clinical investigation. The wastewater samples including the sludge is highly heterogeneous along with the clinically relevant virus load is minimal, specifically log phase of the infection spead, the suggested methodology requires the screening and isolation of viral components from the heterogeneous mixture followed by purification and complete recovery including molecular analyses with highly efficient and sensitive approach. Hence, technical improvements are required better efficiency in terms of virus screening as well as analytical study.

Subsequently, quantification methods require improvements for monitoring the infection rate, prevalence or consistent clinical screening of the virus including multiple variables. Several reports based on WBE approach conducted controlled experiments to closely determine SARS-CoV-2 prevalence and regulation by screening the limiting factors associated with RT-qPCR. Many studies associated with the WBE approach for the screening of the virus lack important experiments for the virus recovery required for determining the estimation and collection efficiency, virus load, and virus genome extraction. Hence, improved technical systems with standardized protocols are required for specifically WBE based detection of the virus RNA.

Currently, majority of the approaches are suitable to test the viruses lacking envelope compared to the analyses of enveloped viruses in the water [52,64]. The COVID-19 virus has been significantly screened in the wastewater with respect to significant concentration along with the application of ultrafiltration, polyethylene glycol (PEG) precipitation, electronegative membrane adsorption, or combinations of these. On comparison with WBE, it still requires technical improvement in order to determine the threshold concentration of virus RNA in the clinical sample when compared with the incidence of COVID-19 infection and thus define the severity of outbreak.

We still lack much clinical information regarding the susceptibility of SARS-CoV-2 infection through wastewater. Clinical evidences are crucial to highlight the significant role of limiting elements responsible for viral transmission, particularly places with poor sanitation [92,93]. Multiple evidences have supported the importance of virus RNA surveillance as an applicable method for the detection of pathogenic variants and track the patterns of viral transmission. In addition, variants are also evolving with new mutations which can further lead to re-emergence of pathogenic variants. A state of emergence as well as re-emergence along with genetic shift in the viruses requires an urgent method to specifically detect the strain of concern from a mixed pool of wastewater samples. We need to perform more clinical as well as fundamental studies to elucidate the paradigm of wastewater based virus monitoring and COVID-19 transmission which can correspond to early preventive measures to restrict the outbreak of enveloped viruses. The study should include variables like diverse transmission dynamics, multiple geographic areas and different solids collection methods at wastewater treatment facilities. Also, it will be relevant to compare influent and sludge from the specific treatment plant to determine and establish the efficient and sensitive approach to reduce the infection load in a particular community or population.

Moreover, studies are required for developing standardized methods to evaluate the load of enveloped viruses like SARS-CoV-2 from the wastewater samples to understand the pattern of virus infection and spread in a population. In order to overcome the limitations of SARS-CoV-2 screening in wastewater multiple mathematical models have been studied and analyzed. One such method is based on flow fluctuations at the inlet of wastewater treatment plant and Kalman smoothing with RT-qPCR screening [114]. Further, SIER model approach was applied to study the SARS-CoV-2 dynamics with respect to infection and disease transmission [115].A mathematical model was applied with population based transmission and virus concentration in the wastewater for studying trend in the virus infection [116]. Li et al., 2022 applied longitudinal monitoring approach for determining the SARS-CoV-2 infection and transmission dynamics [117]. Further, techniques are required to develop machine learning and AI programs to determine the significant association between virus load and rate of positive infection cases in a marked locality or community. But, sufficient reports are still required for successful application of WBE. However, sampling wastewater even in the absence of preliminary information based on the evidences of positive cases or infection shift the technique is still applicable to examine the association of virus infection with the waves of COVID-19. The sample screening from wastewater alo requires standard protocols with respect to SARS-CoV-2 detection and analyses. In the mean time of advancing the WBE approach, tested for sampling from the wastewater can be performed as a practice model for the surveillance of the infection. Studies have also highlighted the significance of overall environment monitoring along with up scaled network of laboratories for clinical testing to prioritize the health based requirements of middle- and low-income countries.

In terms of health management, wastewater epidemiology has emerged as a comprehensive, low-cost, economical, and rapid method for assessing the health condition of the infected population both quantitatively and qualitatively. The WBE method is quite effective in consolidating clinical samples that comprise the health status against the infection transmission in local population groups across the world. Due to the difference in wastewater treatment ranging from developing to developed countries, the WBE approach could be beneficial in monitoring underprivileged geographical zones, which lack proper health assessment facilities and are unaffordability to the major population of people. However, the potential of wastewater in determining the coronavirus spread still needs multiple routine sampling, but the representative data from various studies can be applied as a baseline to monitor the virus load, and track the public health data comprising a broad range of health complications and infection recurrence threats. Notably, the developed countries have applied the WBE system to incorporate the clinical data covering most of the intrinsic and extrinsic parameters for assessing the health status and virus infection. But, the limitations or poor infrastructure of wastewater treatment restricts the clinical investigations required to gather information and take prior action to prevent the further spread of the virus. Further, wastewater treatment facilities are a structural part of maintaining the ecosystem as well as health conditions. Thus, wastewater-based epidemiology can provide an alternative way in terms of health and disease management comprising low to high-income countries around the globe.

2. Conclusion

Wastewater-based epidemiology of SARS-CoV-2 implementation is advantageous on the basis of infection monitoring and spread control on a community scale. The WBE approach in monitoring the viral load will be helpful in following the dynamics of virus progression and ensuring timely response to prevent further spread of the virus in a particular containment zone and/or globally. With limitations, implementation of WBE in indistinct sewage systems lacking centralized facilities for sewage treatment and monitoring would be an obstacle in determining the viral load. However, improving the system with current techniques and equipment as per the WBE approach of screening might shed light on assessing the early identification and monitoring of SARS-CoV-2. Hence, the application of WBE can prove to be advantageous in terms of SARS-CoV-2 detection, monitoring, and prevention based on geographical location and/or globally.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr. Jayavel Sridhar reports financial support from RUSA phase 2.0 for sponsoring “Centre of Excellences in Computational Intelligence and Data Analytics”. JS also thank Madurai Kamaraj University for providing infrastructure. Rahul Parit reports a relationship with Madurai Kamaraj University that includes: non-financial support. Govindaraju Boopalakrishnan reports a relationship with Madurai Kamaraj University that includes: non-financial support. Johni Rexliene reports a relationship with Madurai Kamaraj University that includes: non-financial support. Researchers previously worked in Madurai Kamaraj University for Ph.D and waiting for defense. Rajkumar Praveen reports a relationship with Madurai Kamaraj University that includes: non-financial support. Viswanathan Balaji reports a relationship with Madurai Kamaraj University that includes: non-financial support.