(se Stadies in Chemical and Environmental Engineering 2 (2020) 100042 Contents lists available at ScienceDicect aero Case Studies in Chemical and Environmental Engineering ELSEVIER, journal homepage: ww. cditorialmenager.com/esceeldefaull.aspx Wastewater-Based Epidemiology to monitor COVID-19 outbreak: Present ® and future diagnostic methods to be in your radar Damia Barcelé ** + pene of ramen Chen, ine of Era Asan nd Wat Rac (DAEA CSIC) Jon Gow 1826, 0804, ero, Spin Camm nc War Ree Cc Tei de a ney of Cr is ra 11 es 120, 1700, Gre. pan Cag of Enron and Rare Ss, AMF Umer Hh 31.0, Ce Rowen The Wi has dened he GID. dein ay i, B20. Ws as nae lon of ele Were eerily sore nj fe" months. Shor aera the Nana Medial Pros Admnraton NMA) SARS-CoV. ‘announced nucleic acid testing as the gold standard for virus detection. Antibody testing Is used as well as a cums ‘opemenary tt for upected ues where nila detection wastegate. ns, mile a bed — fotymerse chin secon (PC) thems detection metho ow lise wel oo he ce) ttreton of SARS Gov in wastewater Ft at cll ara the lobe mee reported inthe lt fw ont ng part f the ole Wastewater Bae Epidemiology (BE) arse. Seton of encetaton Inthe and primer, abrtoryltercompraon and varus moles of PCR detonate vi In Comper water rie er lagged pan blest ei gent invent Novel eect treaance srt, sped dota realned vis detection wl be dacs ere swe This it Sopris davies min efor nal pros ike Chere Req Interpol Short Pani Re pest (RSPR, gt PC, Laon (10) an elated plans ela sess The st pat wl be eve the etifetion fmol to target Cori 19 cea based on eatery espns Wo tarts en the. To his cad thi open paper bing for icon the fae of CR tons Tito ar wel a nm dagretc meds in BE 1. Introduction Another key step inthe PCR detemination are the concentration methods. There isa variety of them in wastewaters. In one of the US Wiastewater-based epidemiology (WBE) can be used as early detec: tion system and to determine the scale of COVID-19 outbreak [1,21 Determination of SARS-CoV-2 is generally carried out with nucleic ‘acid-based polymerase chain reaction (PCR) assay, and used for confi mation of COVID-19 patients around the globe. PCR determinations do offer high sensitivity and specificity, but require complicated sample handling in the laboratory, skilled personnel, and along period of dat processing and analysis (4-6), Vist data on COVID-19 in sewage using PCR were reported for a variety of countries, among them the Netherlands [3], Taly [4,5], Australia [6], Spain [7] France [8], Japan (1, USA [10,11], Beuador (12) india (13) and Germany [14]. This PCR methodology relies on the fact that an average of 15-82% of patients fnfcted with SARS-CoV.2 have detectable vial RNA in feces, even in the absence of gastrointestinal symptoms or diarrhea [15]. Samples may ‘continue to remain positive in the stool, even when respiratory tract, samples become negative whereas urine i often negative. ‘mal alee dbeqam@idesiec hups://doLorg/10:1016 eso 2020100082 studies [10] ultrafiltration and adsorptionelution in electronegative ‘membranes were used for wastewater concentration. It pointed out that ro one ofthe secondary and final treated eluents samples with chlorine were tested positive for SARS-CoV-2. The second US [11] study from “Massachusetts did se a different concentration method based on unl: tered sewage precipitated with polyethylene glycol 8000 (PEG). Remarkably, vial iters observed were significantly higher than expected based on clinical eases, being atrbuted to asymptomatic patients not ‘measured in clinical tests. In Italy [5] SARS-CoV.2 RNA was detected in aw, but not in treated wastewaters (four and two samples, respectively, sampled in two dates) Viral RNA was not detected in the Milano and Monza wastewater treatment plants (WWTPS), equipped with tertiary treatments, being in agreement with the case of the Murcia region (Spain) using as well tertiary treatments (7). Interesting to remark that levels of SARS-CoV-2 detected in urban rivers of Quito receiving direct discharges of wastewaters are similar to those of Valencia and Pais, with Received 29 July 2020; Received in reise form 9 September 2020; Accepted 10 September 2020, 2666 0164/0 2020 The Author(s), Published by ever Lid Thisis an open access article under the CC BY-NC-ND license (hp://reatinecommons.og/licenses/y cate)'5000-10000 hospitalized cases. The main difference was that in Quito 1121 only 750 Covid-19 clinical eases were reported, suggesting a lack of PCR-based diagnosis, With this said, the fist explanation could be elated to the faet that Eeuador is a low sanitation country, with not many fi ‘tiesto perform the clinical tests, Another point could be attributed toa high level of asymptomatic eases, as reported in one ofthe US eases [11]. “Another relevant example ofa ist study in large country comes From India (12]. WBE surveillance with reverse transcription (RT)-qPCR was Performed at the Old Pirana WWTP at Ahmedabad, Gujarat, which has 4106 million liters per day and receives effluent from Civil Hospital treating COVID-19 patients. Several genes of SARS-CoV-2 were detected ‘only inthe influent with no genes detected in effluent. Increasing levels ‘of SARS-CoV-2 genetic loading in the wastewater did correspond to an increase in the number of aetive COVID-19 patients in the city. The umber of gene copies was comparable to those reported in untreated wastewaters of Australia [6]. Lastly, the most recent example is fom Germany {14]. Nine municipal WWPs from different cites of the Fed feral State of North Rhine-Westphalia were sampled. A set of 'SARS.CaV-2specifie genes, as well as pan-genorypie gene sequences ‘covering ther coronavirus types, were detected using RT-qPCR. To this end it should be pointed out that other ways to measure ‘COVID-19 outbreak were already highlighted in two opinion papers [1,21 such as rapid! ELISA/biosensors/Paper-based tests and) monitoring of ‘exposure biomarkers. Paper-based deviees would be certainly one ofthe best measurement solutions for the rapid and on-site detection of ‘COVID-19 in sewage waters and humans as well [2,16] and also the use of other biomarkers of exposure 1]. Recent iterature repored the use of Iab-ona-chip (106) procedure [17] to analyze SARS-Cov-2 outbreak mainly for elinical purposes In shor, itis obvious that qPCR isthe method of choice in mast ofthe laboratories involved in WBE to detect SARS.CoV-2. This opinion paper wants to bring attention tothe reader first that PCR methods should be Improved in terms of comparability nd sensitivity when applied to WBE. ‘mainly due tothe complexity ofthe wastevtater matrix, Most importantly isto encourage the scientists involved in virus detection in WBE to think ‘outside the PCR box by considering other complementary ways to detect, ‘COVID-19 outbreak in wastewaters. 12. Direct virus concentration and detection methods- PCR based. Detection of SARS-CoV-2 in sewage has been employed as a com: plementary method to clinical test tf an early warning indicator of virus spreading in communities, covering both symptomatic and asymptomatic eases. As already mentioned, RT-PCR analysis isthe most ‘commonly used method to determine the concentration of Viral RNA in wastewater. To notice that not only PCR detection is relevant when analyzing wastewaters, but concentration methods and the RNA extrac: Lon protocol are Key steps of this methodology. Generally similar pro: tocols forthe influent, secondary-reated sewage are used and a volume ‘of 100 mi. of untreated wastewater simples i sufficient to detect enteric Viruses. Most of the methods developed were used for non enveloped ‘enteric viruses such as norovinas, and hepatitis A virus. There is a ‘comprehensive list of methods for concentrating viruses from wastewater like electropositive or electronegative membranes, ultrafiltration, poly ‘ethylene glycol (PEG) precipitation, ultracentrifugation, skimmed -milk ‘occulaion, monolithic adsorption filtration columns among others Excellent review papers on concentration methods of viruses from wastewater were recently published (15,1822). D Lu [21] did highlight that PEG-based separation method isthe most used for the COVID-19 in WBE. The authors indicated as well that the electronegative membrane filtration method may have problems with the preferential adsorption of ‘organic matteron the charged membrane surface and the potential risk of ‘logging when handling turbid wastewater samples. Standardization of the analytical protocols for determining SAR-CoV-, 2 in wastewaters by different PCR platforms is as well a matter of ‘concern. To investigate a bit more inthis direction De La Rasa and co Sie x Chale rom garg 2 (2020) 100002 workers [ 4 ] did analyze the presence of SARS-CoV 2 using three diferent nested RT-PCR assays and one real-time qPCR assay. Primers were also indicated to be very relevant using this methodology. From al the different methods used. a novel nested PGR assay specific for SARS- (CoV-2 detection was chosen, ‘As drawbacks it should be indicated that important information on the analytical approach is often lacking, while there is still no optimi- zation of the whole protocol sampling, sample storage and concentra tion, RNA extraction and detection/quantifiation. A recent critical review paper [20] identified the main isues for consideration, i, the development of validated methodological protocols forthe virus qua: ‘ative analysis in WBE. The last review on this topic from MVA Coxpu Cal. (22) highlights the efforts to improve efficiency of virus detection and quantification methods in the complex wastewater and sludge ‘Novel approaches based on RNA detection like CRISPR and Digital CR (PCR were used for elinial and aquatic environment applications (21-23). GRISPR is a powerful technology, mainly employed in gene editing. CRISPR is based on RNA detection and it ean achieve low level detection within 20-40 min. A low-cost and accurate CRISPR-Cas12 based lateral ow assay for detection of SARS-CoV-2 was already re- ported [25] The entre time of this assay is less than 40 min. In contrast, RT-qPCR needs 4b. In recent years, dPCR has gained attention as a novel approach to detect and quantify nucleic acids (24,25). The major benefit of dPCR over qPCR is the direct absolute quantification of virus genome copy ‘umber in a sample without the necessity of external calibration. dPCR platforms can generally be divided into two groups: droplet dPGR (emulsion based) and chip-based dPCR (microfuidie). The limit of detection of PCR is atleast 10-fold lower than that of RTE-qPCR. The major advantage of GPCR over qPCR is that it performs absolute quan- tification, and hence, no standards are required. 3, Lab-onachip (L00) and related platforms ‘An emerging field of microfluidies also known as the lab-on -chip (LOC) technologies oF micro total analysis system includes a wide range of diagnostic devices. LOC technologies advanced fom original devices that can conduct a single tsk to integrated systems capable of performing complex jobs. Each integrated LOC platform typically con- tains ses of microfluidic elements, dedicated to single operations such as reagent storage, fluid transport and mixing, detection, and possibly collection. Currently, the systems consist of complex devices with inter: connected micro-channel networks provided of, valves, mixers, PUpS, reaction chambers, and detectors. In short, they are able to perform many laborious benehtop protacols with minimal operator handling in elinial diagnostics and with a low-cost mass prdiction. LOC based techniques ‘ce widely used for vital detection in clinical applications lke inthe cases Of Ebola virus disease, dengue fever hepatitis and human immunodef clency virus (HIV) and others (25 28}. The LOC concept has been expanded using different ypes of configuration, called LionX systems, adopting miniaturized fluidie manipwlation platforms and automated Virus detection. AS an example LOC-related platforms are depicted in Fig. 1 and include Lab-ina Tube, Lab-in-a Box and even Lab or-2-Drone systems among others (28). PCR systems for Point of Care (POC) oF POC testing (POCT) contributed to diagnosis during pandemics such asthe 2014 Ebola virus ‘outbreak. In this respect, Bill & Melinda Gates Foundation, developed the CGeneXpert Ebola Assay based on the GeneXpert system. The GeneXpert Ebola Assay is fully automated and only requires the plaing ofthe pa tient sample into the eartidge and inserting the cartridge into a compact desitop-level instrament. Ietakes ~2 h fr an entice assay. The COVID-19 tlobal pandemic has greatly peed up the development of POCT systems, as wel asthe numberof companies prepared to fund such work. There is along list of companies that have developed systems forthe diagnosis of CCOVID-19-ike outbreaks () Eiken Chemical Co, in Japan based! onmultiple testing using a microfluidic cartridge with 25 wells, (i) Roche ‘reated a POCT system called cobast Liat, a fast and fully automated sampleo-answver system based on a PCR capable of testing samples in 20 min or les.) ID NOW, from Abbott Laboratories i a fully inte- ‘grated sampleo-answer that is currently. available with modified primers to diagnase the COVID-19 vius,(v) Riofirow Filmarrays, fom BioMericus, uses microfluidic technology integrating nucleic acid ‘extraction, purification and PCR amplification into a single chip and resulting in sequential and accurate detection, It was previously used for the detection of Ebola vius and, at present, a COVID-19 test kithas been approved by the FDA (\). Genexpert® developed by Cepheid in tegrates sample preparation, nucleic acid amplification and detection {no a small detection kit (vi) RIsochips proposed by CapitaBio™ in ‘China ean detect 6 common respiratory viruses including COVID-19 in a single chip within 1.5 h. This system not only detects SARS-CoV-2, but also effectively identifies patients with influenza and COVID-19. Compared to the SARS-CoV and MERS-CoV outbreaks, LOC has played a crucial role in the COVID-19 outbreak. Countries including the USA, China and Japan have approved the use ofthis technology, which fully demonstrates the application value of LOC. Main advantage is that most of these technologies ean be used without professional skis opefally at certain moment applications to detect SARS-CoV-2 and, ‘other viruses in wastewater will be developed based on these LOG/POCT systems that will enable simple, fast and sensitive virus detection. Such technology is generally equipped for both fiome an clinical use. 4. Biosensors ‘The global risk of viral disease outbreaks emphasizes the need for rapid, accurate, and sensitive detection techniques to speed up di agnostics allowing cary intervention (2,29-23]. Biosensors are simple ‘and costetfective smart sensing systems for rapid, high-sensitivity ‘defection with an integrated sample preparation and flexible to detect, «ferent targets using the same platform (such as CRISPR- powered sys- tems), and able of simultaneous decetion of different analytes ‘Main types of biosensor used for clinical applications are based on electrochemical reactions (EC), surface enhanced Raman scattering ig- 1. The Lionk Systems for NA Based Vis Detetion (Reproduced with permission from Quin et ly 2020 (128), (SERS), eld effect transistor (FET), or surface plasmon resonance (SPR) detection. Genosensors and immunosensors are also usd for this pt pose. Fewr applications of biosensors such those based on SERS (ig. 2) and FET (Pg. 8) wil be reported in more detall below. ‘A Raman seattering-based lateral low immunoassay (SERS-based LEIA) stip for simultaneous detection of influenza A HINT virus and human adenovirus (HAV), that uses Fe304@Ag. nanoparticles, was recently reported [32] (see Fg, 2). This ystem allows specific recogn tion and magnetic enrichment of target viruses in the solution and SERS detection ofthe viruses onthe stip. Based on this strategy, the magnetic SERS strip can direetly be used for the analyses of biological samples without any sample pretrestment steps. Further, this strip is easy to operate, rapid, stable, and can achieve high throughput EC biosensors have been widely used to detect nucleic acids, proteins, Fig. 2. (a) Symbetle Route for Anubody-Nodiied FeS04@Ag Magnetic Tags and (b) Schematle Digeam of the Magnetic SERS Suip for Detetng Two Respiratory Vinseseproduced with permission from Chongwen Wang ta 2019 (32)Fig. 3. Schematic diagram of COVID-19 FET sensor operation procedure. Gra pene as sensing material i selected! and SARS-CoV-2 spike antbedyiscon ‘ged onto the graphene shet via 1-pyenebutyrie aid a hydoxysueinimide ‘eter, which san icertacing molecule se asa probe linker. Reproduced with permission from (Seo e¢ ab, 2020 (1, ‘small molecular antibodies, and vieuses. The work of Seo etal [S1}re- ported a FET biosensor for detecting SARS-CoV-2 in clinical samples (see Fig. 8). This sensor was produced by coating the gate of the transistor made up of graphene sheets, with an antibody that was specific against the SARS-CoV’2 spike protein. Desired performance of the sensor vias Identified with tests conducted with the SARS-CoV-2 spike protein an- Uigen, cultured virus, and nasal swab specimens ftom COVID-19 patients. ‘This biosensor was further used for the successful detection of viral, strains in eulture medium. ‘One of the questions when working with new technological deviees, like biosensors isto know the limits af detection (LODs). Since this paper targets an environmental audience, the readers may not be familiar with the terminology used in biosensors applied to virus detection. One ofthe ‘most commonly wed measures to express the LOD isthe Plaque Forming Units (PFU)/m. or yl. PFU is a measure used to deseribe the number of ‘virus particles capable of forming plaques per unit volume. Generally i ‘expressed as PFU/ml or PFU/i- and indicates how much viruses infect target cell Inthe case of SERS (sce Fig. 2) LODs for Lass fever, West Nile Fever and Influenza were 230, 195 and 10 PFU/uL respectively. As regards to PET-biosensor devices (se Fi 2) LODs for SARS-CoV-2 are in the range of 16 PFU/ml or 16 = 10-* PFU. In short, sensitivity with FET-biosensor is much better than with SERS-biosensos. ‘The main question here is, similarly as in the case of LOC/POG, ifany ‘of these dvices ean be easily switched 0 also determine viruses in wastewaters, A couple of recent works [16,32] reported the possible application of paper-based sensor devices to virus detection in WBE approach. ‘The first one [16] reports few examples on the use of paper-based biosensors as potential biosensor devices to track like influenza, HIV viruses among others fn wastewaters. The results of the second paper [35] were further eross validated with a robust electro: phoresis and agarose gel image assay, showing promising reliability for wastewater analysis, [As far as Tam aware a paper-based device to detect SARS-CoV-2 in wastewaters is not yet available. The problem is that wastewoters representa very complex matrix, even more than human flu and most probably some kind of extensive clean up to remove the matrix in- terferences will be required prior to biosensor detection. In short, biosensor should be a fast “sample-to-answer” analysis, method which ean provide quantitative monitoring of nucleic acd and ‘genetic information through the analysis of sewage. The propased bio sensors should show advantages including alfordabiity, rapid analysis time, good sensitivity, specificity, and low reagent/sample consumption. 5. Biomarkers Biomarkers must be discharged through urine and feces and need to be specific to human metabolism. Biomarkers re of interest because they Sie x Chale rom garg 2 (2020) 100002 can be rather specific for given infectious diseases and obviously can be ‘wed for WBE, OG Daughton [3] did write a lot about biomarkers of endogenous human biochemical processes, Such biomarkers contin ously undergo urinary or fecal excretion and represent the sum total contribution forthe real-time population served by any given sewage system. One of the first examples used of a possible biomarker for pop lation estimation was coprostanol, which is the predominant reduced sterol formed in the human gut. Several other endogenous biomarkers of Positive health versus disease were also reported by Ch Daughton in another paper [35] lke isoprostanes, dlesmosines, bone turnover markers (Bmp, polyamines and monoeyte chemoattractant protein (MCP) among others. ‘With this said, WBE could target endogenous biomarkers that are significantly elevated inthe diseased state, like in the ease of COVID-19 tutreak and excreted extensively in urine. A good stare to evaluate the tse of biomarkers would be to make use ofthe fact that COVID-19 can involve remarkable inflammatory damage. A well-known biomarker for oxidative stress isthe prostaglandin-like class of substances called is0- prostanes [1,6]. The cantrol of inflammatory response biomarkers for the SARS-CoV-2 infection were recommended in clinical studies in the ‘Zhejiang University School of Medicine in China and the list includes: reactive protein, procalcitonin, ferritin, Dlimer, interleukins IL-4, 16, 1-10 and interferons (FN) (37). SARS-CoV.2 nucleocapsid protein was cently characterized and identified fr diagnostic purposes a the First Aliated Hospital of USTC, University of Science and Teck: nology, of China, in Hef, Anhul in China [98], Recently our group was involved in the proteomic identification of large molecules in WWTPs being identified as disease biomarkers [99]. Such type of approach ‘maybe useful for monitoring changesin the proteomic profile of diferent populations to better understand the sale af new epidemiological threats like COVID-19, ‘Analysis of biomarkers might have several other major advantages, over the use of PCR basically on the detection side since most of the measurements for biomarkers molecules are carried out by mas spec trometry (MS) or ELISA in contrast to PCR. It is well-known that MS or ELISA provide better accuracy and detection limits and validation of results as compared to standard PCR. Other possible ways to target SARS-CoV-2 could be via different biomolecules or biomarkers in sewage that have not been yet described and were only indicated for clinical purposes. For the COVID-19 testing, apart from the most frequently used viral RNA, novel coronavirus ex hibits spike proteins which are immunogenic hence, immune system is able to produce immunoglobulins to trigger an immune response against the pathogen. Importantly, these immunoglobulins are not only valuable {o detect COVID-19. Immunoglobulin M (Ig) antibodies ae produced during the onset of the infectious disease (between 4 and 10 da), whereas immunoglobulin G (lgG) response is produced later (around 2 weeks) ‘With that being sai, it maybe possible to explore the possiblities to Took for the same type of biomarkers, ie inflammatory response bio- markers already used in clinical diagnostis forthe early detection of CCOVID-19 outbreak in WBE. Environmental proteomics seems to be the right tol that can do this job timely and with comprehensive comple ‘mentary information to PCR-based methods. 6. Conclusions and recommendations PCR platforms like RT-qPCR are stil the most widely used methods for SARS-Cov-2 detection in waste waters. As reported earlier, one ofthe problems isthe complexity ofthe wastewater matrix that needs 10 be treated and cleaned up by using different concentration methods. ‘There isan urgent need as well to evaluate RT-qPCR methods used by diferent laboratories with a clear target to achieve a vrification/stan- dardization status (2,19). Several factors such as gPCR platforms, PCR inhibitors, nucleic acid extraction efficiency and low level of targets may have contributed to the observed discrepancies between laboratories. At‘resent most ofthe data reported around the globe as frst detection on ‘SARS.CoV.2 can only say that the virus was detected but in most eases it is impossible to compare the data as vral content among the laboratories in different countries. Their protocols ate diferent and require stan ‘dardization such as concentration method, PCR assay, and process con trols. In addition, the large uncertainty in the viral load in feces makes it ficult to determine atypical value that could be useful in WBE and for ‘comparison with clinical data of the COVID-19 patients, including asymptomatic ones. ‘With tis said, the expected future of PCR in WBE needs to incorpo: rate new technological developments from the elinial field such as ligita PCR, CSRPP, LOC/POC and FET or SERS biosensors among others. ‘Such technological devices should stl be adapted to WBE and need to be ‘economical, portable, and user-friendly. Sewage sensors, such as paper based and smartphones for SARS-CoV-2 detection at the popilation level have a5 well a clear potential for early warning of COVID-19 pandemic. Nowadays it i possible to use smariphone-based biosensors targeting antibody/antigen targets as home POC. technology. Can smartphone technology be used for detecting viruses in wastewaters? “This will need certainly further developments to adapt technologies used ln clinical laboratories like LOC/POC and biosensors to WBE. This has bbeen a common problem inthe field of new biosensors technologies for wastewater messrements. One ofthe reasons for this i that the envi Fonmental market is too smal as compared tothe clinical one to be able to go to mass production biosensor units for WWTPs worldwide. But not ‘only in the detection side technologies need tobe implemented. In short, | WWTP plays a key roe to improve virus transmission. Based on analogies ith previous stulies on SARS and MERS outbreaks, here ae reasons to ‘conclide that the viral content may be somewhat controlled depending ‘on the treatment technology used at WWTPs facilities. Hopefully water utes and water authorities will push together inthis direction to develop biosensors to detect early outbreaks of COVID-19 or any other new virus that may come in the future as well as to improve WWTP technologies ‘To this end the solution at present to monitor COVID-19 outbreak in WBE could be a combination of technologies and methodological ste tegies already in place such as PCR technologies and endogenous bio ‘markers measurements using ELISA and or MS. Why not using both approaches for WBE? That would help to tackle the problems in a more ‘comprehensive and professional way. Certainly many discrepancies ‘observed up tll now could be solved. Hoth measurement methods got advantages and disidvantages. For biomarkers the chemical measure. ‘ments are accurate and sensitive but the main question is that most of biomarkers are not specific ofa given disease. For instance inflammatory response biomarkers are obviously related to SARS-CoV-2 but also to ‘other diseases. But an advantage in ease of the present pandemic situa- tion would be thatthe majority ofthese biomarkers will be related to COVID-19 patients. Lastly, WBE seems to detcet more possible eases of patients, including asymptomatic ones and also other anes who did recover form COVID-19. In this sense WBE can provide additional in- formation not only on asymptomatic eases but also on immunized pz tients who did recover from COVID-19. Based on these data, epidemiologists could be able to estimate if COVID-19 outbreak would become like @ common fi inthe years to come. Declaration of competing interest ‘The authors declare that they have no known competing financial Interests or personal relationships that could have appeared to fluence the work reported inthis paper. ‘Acknowledgements ‘The author kindly thanks the help of Dr Bozo Zonja, Uppsala Uni versity, Sweden, for managing and depicting Figs. 1-3. Se x Chal an rom gag 2 (2020) 100002 References {a} 6. Dough, Wastewater svelte for populatn-witeCOVID1 The prem and forte, Seal ron, 73 0), 19062, [2] D Baro, An environmental and eats pnp frCOMID 19 outa: ‘ete a uly fr, ae spy nic op {Chomal Engine 8 (202), 04006, [a] G Mena Leo, gs elmer, A Beaver Presence of SARS (Gos) S116 [4] © ta Ron Mt Ice P. Mand G Benton Feo Ve Bondo, fe ay Se Total Eten. 736 (020), 139652 15] SC. Rime Sta A. Giga oes, F-Conantore, Dn MM. Marea, Langa, A Macon Romer Pagan - Cpl, {Cowl Maja ALE Gon, Fler, Presence al net of SARS (Cov vrs in wasters and eS. 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