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

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.


Introduction
The current ongoing coronavirus disease-2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has wreaked havoc on global economy, businesses, communities, and public health due to widespread infection, with 43.7 million confirmed cases and 1.17 million deaths in 218 countries as of March 31, 2022 (WHO, COVID-19 Dashboard).Two major obstacles in managing SARS-CoV-2 rapid infections are related to the difficulties in identifying infected people who are asymptomatic for preventing the spread of viral infections in the communities [1,2].Over the past 18 months, the SARS-CoV-2 has diversified through multiple new mutations and 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 latest Omicron (B.1.1.529)and Neocov variants have been discovered and propagated into different parts of the world as variants of interest and concern [3e5].These altered genetic factors increased transmissibility, virulence, disease severity, and mortalities while they also decreased the effectiveness of current therapeutics and vaccines [6e8].
Since the beginning of the COVID-19 pandemic, the standard clinical diagnostic testing (CDT) has been a recognized, valid system for monitoring infectious diseases like 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 the world [10e12] 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 very crucial to establish a unified system incorporating CDT and WBEM to identify infected individuals with COVID-19 hotspots while discovering new variants and mutations, monitoring the current pandemic scenario, followed by anticipating future waves [15].CDT and WBEM can be used synergistically to track local COVID-19 epidemics where clinical samples will be used to identify the SARS-CoV-2 symptomatic patients and WBEM will be optimized as 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) [16e20].As a result, adapting the collective approach combining WBEM 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 doses 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, adopting 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 7e14 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 combining both CDT and WBEM for symptomatic, asymptomatic, and paucisymptomatic carriers would allow early detection of new variants, and be of potential help for advancing the process of vaccine development.From our recently 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 [118], the number of SARS-CoV-2 positive patients were lowest when the CT value was high (lowest gene copy number) in wastewater samples.On the other hand, when the number of positive patients increased the corresponding CT value was low (the highest copy number) in sewage samples as detected in the same study.In addition, increased signals of the SARS-CoV-2 genetic biomarkers were noticed earlier in WS compared to the viral load in clinical samples of the positive patients.
There are limited studies that link the concentration 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 [31e33].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 clinical testing.This review emphasizes that the combined monitoring of SARS-CoV-2 using CDT and WBE systems can guide the way forward for effective surveillance of the prevalence of infectious disease such as COVID-19.

Clinical diagnostic test (CDT) and Wastewater-based Epidemiologic Monitoring (WBEM)
The accurate and rapid clinical diagnostic 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.The clinical signs and symptoms of a patient such as fever, dry cough, headache, and shortness of breath usually develop 2e14 days after the exposure to 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 cost, laboratory facilities with proper instruments, expert hands, and availability issues [81,120,121].
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 the eradication of poliovirus, and this is recognized as the first application of wastewater-based epidemiological (WBE) investigations [39].The SARS-CoV-2 genetic markers have been identified in feces from presymptomatic persons even 1e5 days before the positive clinical test [29,30] and in people with mild signs and symptoms [41].Recent investigations of a few WBE studies have detected COVID-19 patients before the onset of clinical symptoms from feces samples, and 48e67% of diseased people had SARS-CoV-2 viral RNA in their stool, which survives in wastewater and can last up to >33 days [41e44].Previous findings showed an association between wastewater viral concentration and COVID-19 confirmed cases where SARS-CoV-2 viral RNA were between 2.0 and 6.0 log 10 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 to be reasonably good in many studies [35e38].Betancourt et al. [46] found that WBEM had 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, the prevalence of SARS-CoV-2 RNA biomarkers in stool was higher (48.1%) as compared to the swab samples of the 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 [45,47,118].
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 the COVID-19 pandemic is that in 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 interpretation of results.The selection of WS collection points plays an important role in representing a particular catchment area (wastewater treatment plants, sewer drains, primary networking system or communal watershed, river course, bus stand, and airport) [22].Heat treatment (60 C, 30 min), 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 in the laboratory manually using TRIzol reagent or commercially available Qiagen, Thermo Fisher kits [52].For calculating viral copy number maximum studies have used the equations 1 and 2 [51,53].

Number of infected individuals ¼
Positive, negative, no-template, and extraction controls should be used with standard curve calculation as well as PCR inhibitors should be checked according to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) rules [54].Major roles and drawbacks of both WBEM and CDT are given in Table 1.Considering the previous research outcomes based on WBEM and CDT results with various validated methods and comparisons of the two approaches, we suggest the following for disease burden correlation: i) analysis of the trends in WBEM data and comparison with the clinical diagnostic test data; ii) observing the effectiveness of the interventions with the declining number of patients; and iii) detection of hotspot areas with COVID-19 cases for vaccination and booster doses [55].
One previous study in Massachusetts between March and May 2020 found similar trends of the abundance of SARS-CoV-2 RNA biomarkers 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 h after a 300% or greater rise in detected 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].

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 [65e67].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   2020) claimed that, SARS-CoV-2 in the stool specimen was found significantly elevated than in the serum/blood specimen or nasal swab samples [86].
A recent WBE study conducted in over 40 US cities found that a weekly incidence might not be sufficient to support the interpretation of viral concentration in wastewater [88].Wu et al. [88] recommended that at least two wastewater samples in a week are necessary to ascertain the accuracy while analyzing the COVID-19 trends.In another study, Petala et al. [89] suggested that a weeklybased sampling method for viral quantification with fixed sampling time could be scheduled to understand the day-to-day deviation.In addition, they strongly proposed that wastewater sample test results should be validated with clinical data.WBES could be followed for other emerging and re-emerging viruses to detect hotspots together with the help of CDT [28,34,87,119,120].

Conclusion
WBEM has the potentiality to detect hotspots, identify the prevalence, and predict early warning for various disease.On the other hand, CDT can be used to diagnose positive patients, undertake 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 it in terms the of rapidness, sensitivity, and portability of the analyses to demonstrate it as 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.

¼
No: of RNA Copies per L Contribution of RNA Copies per person to total sewage water ðLÞ

Table 1 A
comparison of the major advantages and drawbacks of CDT and WBEM.

Table 2
Salient examples of integration of wastewater based epidemiologic monitoring with clinical testing data.
Germanysuitability for COVID-19 surveillance and potential transmission risks.Sci Total Environ 2021:141750.15.RakibSH, Masum S, Patwari MRI, Fahima RA, Farhana A, Islam MA: Design and development of a low cost ultraviolet disinfection system to reduce the cross infection of SARS-CoV-2 in ambulances.In International Conference on Elec-Moe CL, Raj SJ, Ong L, Cherles K, Ross AG, Shirin T, Raqib R, Sarker P, Rahman M, et al.: Wastewater surveillance of SARS-CoV-2 in Bangladesh: opportunities and challenges.Curr Opin Environ Sci Health 2022:100334.This published review discussed major challenges and opportunities for wastewatersurveillance in Bangladesh, where they indicated to use clinical data with wastewater based surveillance. .Xiao A, Wu F, Bushman M, Zhang J, Imakaev M, Chai PR, et al.: Metrics to relate COVID-19 wastewater data to clinical testing dynamics.Water Res 2022, 212, 118070.This article highlighted that the time lag and transfer function analysis of the wastewater data preceeded the clinically reported cases in the first wave of the pandemic, but did not serve as a leading indicator in the second wave due to increased testing capacity for case detection and reporting.96.Bertrand I, Challant J, Jeulin H, Hartard C, Mathieu L, Lopez S: Scientific Interest Group Obépine, Schvoerer E, Courtois S, Gantzer C: epidemiological surveillance of SARS-CoV-2 by genome quantification in wastewater applied to a city in the northeast of France: comparison of ultrafiltration-and protein precipitation-based methods.Int J Hyg Environ Health 2021:113692.97.Martin J, Klapsa D, Wilton T, Zambon M, Bentley E, BujakiE, Fritzsche M, Mate R, Majumdar M: Tracking SARS-CoV-2 in sewage: evidence of changes in virus variant predominance during COVID-19 pandemic.Viruses 2020:1144.Presence of SARS-coronavirus-2 RNA in sewage and correlation with reported COVID-19 prevalence in the early stage of the epidemic in The Netherlands [Internet] Environ Sci Technol Lett 2020, 7:511-516.https://pubs.acs.org/doi/10.1021/acs.estlett.0c00357.This paper discussed the strength of wastewater for SARS-CoV-2 surveillance as early warning tool and to predict the number of patients in sewersheds.99.Pillay L, Amoah ID, Deepnarain N, Pillay K, Awolusi OO, Kumari S, Bux F: Monitoring changes in COVID-19 infection using wastewater-based epidemiology: a South African perspective.Sci Total Environ 2021:147273.100.Ahmed W, Tscharke B, Bertsch PM, Bibby K, Bivins A, Choi P, Leah Clarke L, Dwyer J, Edson J, Nguyen TMH, et al.: SARS-CoV-2 RNA monitoring in wastewater as a potential early warning system for COVID-19 transmission in the WBEM and CDT for detecting high-prevalence areas Islam et al. 13 www.sciencedirect.comCurrent Opinion in Environmental Science & Health 2023, 31:100396 community: a temporal case study.Sci Total Environ 2021: 144216.101 * .Gonzalez R, Curtis K, Bivins A, Bibby K, Weir MH, Yetka K, Thompson H, Keeling D, Mitchell J, Gonzalez D: COVID-19 surveillance in Southeastern Virginia using wastewaterbased epidemiology.Water Res 2020:116296.This study also confirmed that clinical patient's numbers can be tracked with the help of wastewater and clinical data.