key: cord-0932381-r7aafsqh
authors: Anand, Uttpal; Adelodun, Bashir; Pivato, Alberto; Suresh, S.; Indari, Omkar; Jakhmola, Shweta; Chandra Jha, Hem; Jha, Pawan Kumar; Tripathi, Vijay; Di Maria, Francesco
title: A review of the presence of SARS-CoV-2 RNA in wastewater and airborne particulates and its use for virus spreading surveillance
date: 2021-02-25
journal: Environ Res
DOI: 10.1016/j.envres.2021.110929
sha: 9f6ab227f90340f43b63026202ac39c6dd739a24
doc_id: 932381
cord_uid: r7aafsqh

According to the WHO, on October 16 2020, the spreading of the SARS-CoV-2, responsible of the COVID-19 pandemic, achieved 235 countries and territories, resulting in more than 39 million confirmed cases and 1.09 million deaths globally. Monitoring of virus outbreak is one of the main activities pursued for limiting the number of infected peoples and decreasing the number of deaths and of patients recovered that causes high pressure on the health care, social and economic systems of different countries. Wastewater based epidemiology (WBE), already adopted for the surveillance of life style and health conditions of communities, shows interesting features for being used also for the monitoring of the COVID-19 diffusion. Together with wastewater, the analysis of airborne particles has been recently suggested as another useful tool for detecting the presence of SARS-CoV-2 in given areas. The present review reports about the status of the art and of the research currently performed concerning the monitoring of SARS-CoV-2 spreading by WBE and airborne particles. The former resulted more investigated, whereas the latter resulted in a very early stage with a limited number of very recent studies. In any case, the main results highlight in both cases the necessity of more research activity for better understating and defining the biomarkers and the related sampling and analysis procedures to be used for this important aim.

The first detection of severe acute respiratory syndrome (SARS) disease caused by SARS-CoV-2 (officially designated by the Coronavirus Study Group of the International Committee on Taxonomy of Viruses) occurred in the city of Wuhan (China) in late December 2019 (CDC, 2020) .

Since that date, the Corona Virus Disease 2019 (COVID-19) has grown rapidly, and exists in 235 countries and territories and have resulted in more than 39 million confirmed cases and 1.09 million deaths globally as of October 16 (WHO, 2020a) . Currently, the most credited even if not fully demonstrated transmission pathway of SARS-CoV-2 virus is the one-based on respiratory droplets, with an average diameter ≥ 5 μm, that could be generated by a sneeze, cough, breath or during normal speaking of infected subjects (Lewis, 2020; National Research Council, 2020; Yu et al., 2018; WHO, 2020b) . However, the presence of the virus within airborne droplet nuclei with a diameter ≤ 5 μm and hence transmissible to distances > 1m is still a matter of debate (Anderson et al., 2020; Lewis, 2020; Morawska and Cao 2020; National Research Council, 2020; Yu et al., 2018;  J o u r n a l P r e -p r o o f detection of virus presence and for the surveillance and monitoring of COVID-19 (Farkas et al., 2020) . WBE was already successfully adopted for the surveillance of life style and health conditions of communities concerning the use and/or abuse of both licit and illicit drug (Choi et al., 2018; Daughton, 2001) . A similar role in COVID-19 detection and management seems to be played also by the monitoring of the airborne particles generated due to both symptomatic and asymptomatic individuals (Mizumoto et al., 2020; Oran and Topol, 2020; . Furthermore, the noninvasive nature of both WBE and airborne particle sampling can also help to prevent any ethical issue of confidentiality and potential stigmatization of infected patients (Choi et al., 2018; Bagcchi, 2020) .

However, the efficient use of this apporach for SARS-CoV-2 monitoring in both water environment and airborne particles requires specific standardization of viral sampling procedures and methodologies along with the appropriate identification of specific biomarkers. The biomarkers are pivotal to providing the required epidemiological information for the early detection of the virus in the environmental compartments of water and air media to forestall further transmission. The present review aims to report about the scientific evidence of SARS-CoV-2 spreading via aerosols and wastewater carriers and on the criticisms concerning the WBE application for an effective monitoring and prevention of the virus outbreak. Particular attention was also focused on the virus transmission and interaction with the host along with the indication of future research needs.

(CoVs) belong to the subfamily Coronavirinae, subdivided into four genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus based on their phylogenetic properties (Cui et al., 2019) .

Betacoronavirus possess the ability to infect mammals. CoVs possess spike-like projections on its surface like a crown, thereby the name. Attributes of the viruses belonging to the family Coronaviridae include the presence of an envelope with a positive-sense single-stranded RNA genome of 26-32kilo bases (Figure 1 ) (Weiss, 2005) . Electron microscopic analysis demonstrated the spherical shape of the virus with an estimated diameter of 60-140nm . In addition, the open reading frame (ORF)1a/b and the remaining ORFs of all coronaviruses encode for 16 non-structural proteins (NSPs) and envelope (E), membrane (M), nucleocapsid (N) and spike (S) proteins which constitute the structural proteins respectively (Figure 1 ) (Su et al., 2016) . into three lineages (i.e., A, B, and C). America and Europe predominantly consisted of the A and C types, respectively (Forster et al., 2020) . The B type was mostly confined to East Asia (Forster et al., 2020) . The complete 29,903-bp-long RNA genome sequence of the SARS-CoV-2 (GenBank:

MN908947.3) contained 5′-capped and 3′-polyadenylated ends and many ORFs .

Notably, the Hemagglutinin-esterase gene, prevalent in lineage A Betacoronaviruses was absent in SARS-CoV-2 . Like its distinct relative SARS-CoV, the SARS-CoV-2 also uses S protein to interact with the host ACE-2 receptor for cellular entry. However, another cell surface protein Neuropilin-1 has also been shown to act as entry factor for SARS-CoV-2. A study based on cryoelectron microscopy elaborated that the SARS-CoV-2 S protein has 20-fold higher affinity towards ACE-2 than SARS-CoV and this is due to 30% difference in the S protein of these two viruses. Unlike SARS-CoV or SARS-CoV-2, the MERS-CoV S protein engages with host dipeptidyl peptidase 4 (DPP4, also known as CD26) for the purpose of entry. The viral protein and host receptor interaction induces conformational changes in the S protein and facilitates entry. After interaction with the host receptor the virus is either endocytosed inside the cell or releases the genome in cytosol through membrane fusion. Once in the cytoplasm, RNA is translated into a polypeptide which is further cleaved by the proteases. Briefly, two viral encoded cysteine proteases; papain-and 3C-like proteases, cleave the polypeptides encoded by ORF1a/b into 15

NSPs . The virus encoded RNA dependent RNA polymerase (RdRp) performs discontinuous transcription and synthesizes sub genomic RNAs. These RNAs further get translated into various viral proteins like S, M, E, N etc. Among these the SARS-CoV-2 E protein, the smallest among all structural proteins, is localized to the endoplasmic reticulum and Golgi complex.

The M protein is the most abundant transmembrane glycoprotein (Nieto-Torres et al., 2011; Siu et al., 2008) and prominently functions in the viral assembly with E and N proteins (Siu et al., 2008) . 

The role that water can play as a vehicle for pathogen spread as a consequence of faecal contamination was historically recognized for several microorganisms and diseases. Among these, the most important viruses such as Norovirus, Enterovirus, Hepatitis A virus, and Adenovirus belong to the Calicivirade, Picornaviridae, and Adenoviridae families (WHO, 2017) . These viruses were detected in several types of water (wastewater, seawater, freshwater, and drink water) and

hence associated with disease outbreaks (Bonadonna and La Rosa, 2019; Gall et al., 2015; La Rosa et al., 2012; Moreira and Bondelind; Rusinol, 2017; Girones, 2017) . SARS-CoV-2 is an enveloped virus belonging to the Betacoronavirus genus in the Coronaviride family characterized by positivesense and single-strained RNA. A significant portion of a patient infected by SARS-CoV-2 showed gastrointestinal symptoms with consequent virus shedding into faeces above those from the respiratory tract Xiao et al., 2020a; . In addition, Xiao et al. (2020a) reported that the virus can infect glandular epithelial cells, and the virions can be secreted from intestinal cells albeit there is limited evidence of the potential infectivity of the virus detected in faecal samples . Faeces was charged to be the main carrier of the virus in wastewater and hence a further potential route of COVID-19 pandemic spreading. As reported in Differently from other microorganisms (e.g., bacteria), viruses showed a very limited survival period without hosting cellules. Furthermore, due to the damage susceptible by the labile lipid envelope, SARS-COV-2 resulted inactivated in the aqueous environment even if this process could result not so rapid. The occurrence of SAR-CoV-2 in municipal wastewater was investigated in the Netherlands by Medema et al. (2020) . Presence of RNA fragments was detected in 6 out of 8 wastewater treatment plants monitored. Similar results were also reported by Wu et al. (2020b) Poliovirus-1 (PV-1). In wastewater at 23 0 C, HCoV resulted inactivated in less than 4 days, whereas PV-1 lasted for about 11 days in primary wastewater and about 6 days in secondary effluents. In tap water, both HCoV and FIPV lasted for about 12 days, whereas at 4°C the persistence was estimated to achieve up to 100 days.

The specific RNA sequences for SARS-CoV-2 reported in the study were detected by qPCR (Foladori et al., 2020) . For improving the efficiency and effectiveness of RNA detection, liquid samples were generally pre-treated for both increasing its purity degree and/or for increasing the virus concentration. By the way, there is a lack of consensus on the protocols and standard procedures to be adopted for these analyses. Among the available methods for the SARS-CoV-2 concentration, proposed a filtration using 0.22 µm membranes followed by PEG precipitation, centrifugation at 12,000 rpm for 2h or until the pellet was visible. Ahmed et al. (2020) proposed two methods: a first method based on direct RNA extraction from the electronegative 0. Nao et al. (2020) . For the same purpose, Randazzo et al. (2020) adopted an RT-qPCR diagnostic panel assay proposed by CDC (2020). All these differences in the procedures adopted leaded to some difficulties in performing direct comparison between the results reported in the studies as the consequent correct and univoque interpretation of the data.

Recently, 239 scientists from 32 countries have written an open letter to the World Health Organization (WHO) arguing the importance of airborne as a transmission route for COVID-19 (Morawska and Milton, 2020) . However, at the moment the airborne mechanisms of transport of the (Milton et al., 2003; Yan et al., 2018; Leung et al., 2020) . Settling rapidity of larger size respiratory droplets (i.e. > 5µm) resulted higher than their evaporation rapidity. Oppositely, the evaporation rapidity of lower size respiratory droplets resulted higher than the settling one leaving residuals called droplet nuclei. These latter may be rich of viruses aggregate and other compounds as mineral salts and proteins (Asadi et al., 2020; Borouiba, 2020) . Due to their dimensions, droplet nuclei can float in the air for long periods potentially contributing to the airborne transmission mechanism (Allen and Marr, 2020; Morawska and Cao, 2020; Martano, 2020) . Air quality also in terms of humidity and temperature was already indicated as another relevant aspect in the COVID-19 spreading (Barcelo, 2020b). Ma et al. (2020) reported during January and February 2020 a decrease of deaths due to SARS-CoV-2 in the city of Wuhan as both air temperature and humidity increased. Opposite findings were reported by Zhou and Xie (2020) for 122 Chinese cities during the same period indicating the absence of evidence between increase in ambient temperature and decrease of infection spreading. By the way, is worthy to be noted that smaller size droplet nuclei can remain suspended in the outdoor air for several days (Brito et al., 2018) before being eventually inhaled by other individuals. This facilitates its dilution below the infective inhalation dose (about 10 5 -10 6 ) but also the inactivation and/or complete degradation of the SARS-CoV-2 and higher temperature can facilitate this process. In any case, low air quality can affect the respiratory apparatus of exposed individuals making them more vulnerable to the virial aggression (Barcelo, 2020b). With specific reference to the role of PM 10 /PM 2.5 as potentially J o u r n a l P r e -p r o o f carriers of infection spreading, the methodological approach adopted in such studies can be classified into two main categories: (a) statistical inference between the concentration of PM and the number of infections and (b) lab analysis of the presence of RNA in the particulate matter.

Bibliography research in Scopus database was performed using the keywords as "COVID-19" or "SARS-COV" or "Coronavirus" and "aerosols" or "droplets" or "PM" or "air pollution" or "air transmission" or "airborne. The research was limited to articles with open access published in 2020 and the English language. Among more than 700 papers, only 6 can be referred to the role of PM as a carrier of COVID-19 and all of them can be referred to the first approach category. Besides, the investigation of the references listed in the most relevant papers led to detect further 3 papers based on the statistical approach and 1 based on lab analysis.

Concerning the "statistical" approach, several studies (Bontempi, 2020; Borro et al., 2020; Chennakesavulu and Reddy, 2020; Coccia, 2020; Delnevo et al., 2020; Fronza et al., 2020; Li et al., 2020; Setti et al. 2020a; found a correlation/association between the PM concentrations (in some cases in terms of number of exceeding of regulated concentration of air quality standards) in several cities in Italy and China. Spearman's correlation coefficient was the most used tool implemented for statistical analysis. However, it remains controversial the causeeffect relationship between infection and PM concentration since as Altman and Krzywinski (2015) explained correctly in their work "Correlation is not causation". Besides, confounding variables are not sufficiently investigated. In the specific case where a mere association or correlation between particulate concentration (variable -X) and the number of infected people (variable -Y) occured, the relationship cannot be interpreted a priori as a cause-effect relationship (X cause Y), because they would overlook other phenomena. For example, the number of infected people could be caused by a third factor (variable -Z) and air pollution would play a role as not the cause but as an amplifier to ("boost") of the virus effects on the respiratory system as some studies have identified (Bontempi, 2020; Comunian et al., 2020; Setti et al. 2020a; . Cities with ≤100 and >100 days with the concentration of PM10 higher than the limits indicated by EU air quality standards, showed an increase in the number of infections of about 0.25% and 85% respectively (Coccia, 2020) . Additionally, Chennakesavulu and Reddy (2020) 

Water-borne diseases such as cholera, diarrhoea, dysentery, typhoid, and polio resulting death rate increase day-by-day globally. It is due to more than 2 billion people using drinking water sources contaminated with faeces in the worldwide (WHO, 2017). Most of these infections are from the water environment through the improper treatment of viruses in the sewage and wastewaters. In the last two decades, SARS-CoV-2 is the third coronavirus to spread worldwide (Guarner, 2020) . As already reported in Table 1 , several studies detected the presence of SARS-CoV-2 in stools of infected patients using as conformity test the rectal swab method (Lamers et al., 2020; Zang et al., 2020) . Moreover, a duration and survival of this virus in such media from 15 day up to 42 days was also reported He et al., 2020; Wölfel et al., 2020) . These The continuously human excreta received from the different sewer systems offer near-real-time outbreak data and these data were containing viruses from infected humans. This can be also related to their symptomatology status like no symptoms, paucisymptomatic or subclinical, and presymptomatic. By the way, it is worthy to mention that Chen et al. (2020) reported about patients with positive faecal specimen but negative pharyngeal and sputum test.

WBE identified viral concentrations in wastewater during different seasonal fluctuations. However, these concentrations were analysed in comparison with other virus protocols. For example, the highest concentration of noroviruses was found during the winter and summer seasons (Nordgren et al., 2009) . Similarly, Li et al. (2011) and Katayama et al. (2008) , reported the concentration levels of rotavirus and norovirus were higher from November to April. This variation in COVID-19 spread also occurs in sewage at locations falling inside of the climatologically favoured zones (4-12 °C) (Poole, 2020) . Due to SARS-CoV-2 is a potentially lethal virus to humans, biosafety guidelines and procedures for concentrating such virus through specific filtration are given by the Center for Disease Control and Prevention (CDC, 2020). Several virus surrogates are used which depends on the experiment type, murine hepatitis virus is used as a SARS-CoV-2 surrogate (Ahmed et al., 2020) in wastewater samples. Transmissible gastroenteritis (TMV) virus, enveloped bacteriophage, non-enveloped Escherichia, and feline infectious peritonitis viruses were used to test the persistence J o u r n a l P r e -p r o o f and disinfection of human coronaviruses in water and wastewater (Boehm et al., 2019; Silverman and Boehm, 2020; De Carvalho et al., 2017; Zielinska and Galik, 2017) . Shirasaki et al. (2017) concluded that plant virus (Pepper mild mottle virus) is a suitable surrogate for the human virus. Lesimple et al. (2020) reviewed recent techniques for virus removal from wastewater like algal ponds, nanomaterials, membranes, and other treatment techniques. They have reviewed these techniques to develop WBE for both sampled (localised infection clusters) and quantified. 

A relevant aspect concerns the selected biomarkers. These have to be a major and exclusive excretion metabolite, chemical or biological product of the specific disease under investigation, characterized by adequate stability in wastewater and the successive sampling and storage process (Sims and Kaspryzk-Hordern, 2020; Zuccato et al., 2008) . In investigating community drug abuse, J o u r n a l P r e -p r o o f Zuccato et al. (2008) excluded from the target biomarkers adopted in their study the most abundant excretion products (i.e., Glucuronic acid-conjugated metabolites) since their high instability in wastewater. Once these two main aspects have been defined, these authors suggested that the wastewater specimen has to be realized by the collection of wastewater samples at the wastewater plant inlet every 20 minutes for 24 hours. This composite sample has to be pooled and performed by an automatic computer-controlling device, in a time-proportional mode for considering the rate fluctuations during different hours of the day. It is also recommended to withdraw more samples on consecutive days for ensuring the correct reproducibility of the tests. Before being analyzed, the samples have to be properly stored for avoiding the degradation of the biomarkers. Maida et al. (2017) suggested filtration the same day of collection and storage at 4°C for a maximum of 2 days.

The analytical procedure differs based on the origin of the biomarkers. In the case of drugs, the targeted biomarkers are urine metabolites and/or the unchanged parents of specific drugs ( Table 2) .

These can be measured by a fully validated procedure (Castiglioni et al., 2006; Zuccato et al., 2008; Maida et al., 2017) consisting in the following main steps: spiking of the water samples with internal standards, acidification and extraction of the solid phase using reverse-phase/cationexchange cartridges. Cartridges were preventively conditioned with methanol, water, and 0.01 N HCl solution. Successively, the liquid eluted obtained by methanol and 2% ammonia in methanol was analyzed by liquid chromatography-tandem mass spectrometry. Quantitative analyses were performed in the selected reaction-monitoring mode by measuring the fragmentation products of the protonated or deprotonated pseudo-molecular ions of each compound and deuterated analog.

Instrumental limits, repeatability, recoveries, and limits of detection have to be also calculated (Castiglioni et al., 2006) . Another validated procedure, with some minor differences to the one proposed by Castiglioni et al. (2006) was proposed by Lai et al. (2011; . Once the concentration of the targeted biomarkers was determined, the whole amounts of biomarkers excreted by the population served by the wastewater treatment plant can be calculated by simply multiplying the concentration by the whole amount of wastewater collected from the plant. Since the population served by the plant was preventively investigated, it is possible to assess the amounts of biomarkers excreted per inhabitant per day (mg/inhabitant/day). The evaluation of the daily consumption of drugs per inhabitant is hence possible by multiplying the amount of mg/inhabitant/day by a corrective factor that represents the percentage of drug dose excreted multiplied by the molar mass ratio of the parent drug/targeted biomarker.

As already discussed in the previous section 4.1.1, also in this case the stability of the biomarkers and the adequate concentrations are important issues able to affect the results of the whole analysis.

If the targeted biomarkers are represented by viral DNA/RNA, the water samples were filtered by nano-ceramic filters eluted for 2 min with 1 L 1.5% w/w beef extract as indicated in EPA's protocol (U.S. EPA, 2001) within 24 hours. The pH of the obtained solution was adjusted to 3.5 ± 0.1. After being flocculated for 30min. and centrifuged at 2500g for 15min at 4°C, the solid phase was resuspended by 30mL of 0.15M sodium phosphate, pH 9.0-9.5, and centrifugated at 7000g for 10 min at 4°C. After centrifugation, the supernatant was firstly neutralized to a pH of about 7.25 and the filtrated using 0.45 -μm and 0.22 -μm syringe filters. Extraction of nucleic acid was performed with 140 mL of purified virus concentrate using a commercial kit. The nucleic acid was stored at -80°C until being further processed. The quantification of the targeted viral DNA/RNA is usually performed by quantitative PCR (qPCR) or reverse transcription (RT) qPCR (McCalla et al., 2020) .

In general, as reported by several authors (Ahmed et al., 2020; Fongaro et al., 2020; Rimoldi et al., 2020; Medema et al., 2020; La Rosa et al., 2020a, b; Trottier et al., 2020; Bar-Or et al., 2020; Randazzo et al., 2020; Kocamemi et al., 2020; Green et al., 2020; Sherchan et al., 2020; Kumar et al., 2020; Haramoto et al., 2020) , more information resulted necessary concerning the survival and persistence of the SARS-CoV-2 in water and wastewater. In any case, its detection and quantification in this media was performed by RT-qPCR method, which is based on the presence of TaqMan detection probe assay are reported by different authors (Wurtzer et al., 2020; Fongaro et al., 2020; Nemudryi et al., 2020; Medema et al., 2020; Bar-Or et al., 2020; Randazzo et al., 2020; Kocamemi et al., 2020; Green et al., 2020; Haramoto et al., 2020) . The choice of this method is based on its reliability due to its specificity and sensitivity properties (Bustin and Nolan, 2020) . Michael-Kordatou et al. (2020) reviewed different methodology protocols for the SARS-CoV-2 quantitative analysis in wastewater. Stachler and Bibby (2014) reported that several viruses were abundantly available, such as pepper mild mottle virus The authors found that the less than 0.8 copies for each size range, indicating less possible transmission through this medium, especially in a less crowded environment. Figure CoV-2 is enabled by patient-generated airborne biomarkers of pathogens found in aerosols and other related volatile compounds, which can be exploited for making point-of-care devices (Bhalla et al., 2020; Gould et al., 2020) . However, highly advanced technology is required to detect the chemical markers from environmental samples such as odor, breath, and aerosolized pathogens like J o u r n a l P r e -p r o o f SARS-CoV-2 generated by the infected patient (Fung and Mykhaylova, 2014; Bhalla et al., 2020) .

While the majority of the studies that were conducted to detect SARS-CoV-2 RNA in either wastewater or aerosol are based on reverse transcription-polymerase chain reaction (RT-PCR) and reverse transcription real-time polymerase chain reaction (RT-qPCR) techniques for qualitative and quantitative information, respectively, and digital PCR (Amoah et al., 2020; Polo et al., 2020; Santarpia et al., 2020; , there have been instances where inconsistent results were obtained (Ahmed et al., 2020a; Rimoldi et al., 2020) . This was due to the diverse contents of the PCR inhibitors and different concentration protocols being adapted to detect the existing molecular markers (Gibson et al., 2012; Hart and Halden, 2020) . On this note, there is a need to identify appropriate biomarkers specific for SARS-CoV-2 detection in the environment, which have been scantly researched.

WBE aims to assess the population's health by the detection and quantification of endogenous and exogenous urinary human biomarkers found in wastewater (i.e. wastewater-based epidemiology) ( Table 2 ). Exogenous biomarkers have been assessed as population markers, whereas endogenous chemicals excreted by human metabolism characterized by low variance have been indicated as those suitable for estimating the population size (Choi et al., 2018) . The use of WBE as a nonintrusive and non-incriminating method for obtaining real-time data truly reflecting the usage of drugs by wide communities was suggested for the first time by Daughton (2001) . In this case, the authors proposed the investigation of domestic sewage for identifying the presence of illicit drugs and synthesis by-products excreted in human's faeces and urine and consequent evaluation of the amount of drug used and of the number of users.

One of the first practical applications of this approach was reported by Zuccato et al. (2005) for estimating the use of cocaine in a broad area of Northern Italy based on wastewater analysis. Based on the detection by mass spectrometry of cocaine and its main urinary metabolite (benzoylecgonine), the authors assessed an average daily use of cocaine of about 27 doses every 1,000 inhabitants. Since that date, the use of this approach for the non-intrusive and wide investigation on the use of both licit and illicit drugs, pesticides, pharmaceutical and personal care products (PPCPs), alcohol consumption, and general health by oxidative stress markers, was largely exploited (Been et al., 2014; Choi et al., 2018; Kankaanpää et al., 2016; Kim et al., 2015; Lai et al., 2016; Reid et al., 2011; Rousis et al., 2017; Ryu et al., 2015) . Since the success has already demonstrated in monitoring the above-described aspects, there is a large potential of using WBE for monitoring also other biomarkers related to infectious diseases (Sims and Kasprzyk-Horden, 2020) . 

Biomarkers are measurable and reproducible biological and chemical indicators containing pathogenic deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA), proteins, inflammation resistance genes, and metabolites emanating from any biological system for assessing the potential environmental risk through its early detection (Sims and Kasprzyk-Hordern, 2020; Strimbu and Tavel, 2010) . Daughton (2012) highlighted examples of biomarker indicators to include human excretion products such as blood, faeces, urine, and other clinical samples. Since SAR-CoV-2 has shown to have similar clinical and genomic characteristics of protein sequence to SAR-CoV , prompting to possible similar biomarkers in the environment (Michael-Kordatou et al., 2020; van Doremalen et al., 2020) . However, recent studies have shown that, unlike the original SARS-CoV, SARS-CoV-2 has multifaceted infections, including respiratory, gastrointestinal tract, nervous system, cardiovascular, and other body organ-related infections .

This implies the production of more viral particles from infected parts, subsequently disseminating into the environment, including wastewater and air through urine, faeces, breath, and possibly odor (Gould et al., 2020; Adelodun et al., 2020a) . Although more studies are still required to conclude on While the majority of these characteristics were used to describe the drug biomarker, the same can be attributed to SARS-CoV-2 since they are both found to exhibit through metabolism and excretion, considering the currently limited data on the pathogenesis of SARS-CoV-2. Sims and

Kasprzyk-Hordern (2020) further noted some desirable characteristics of biomarkers that can be fitted by WBE techniques. The excretion of the biomarker must be through urine with concentration measured in ng/L and high enough to be detected at the downstream of the wastewater. As already J o u r n a l P r e -p r o o f mentioned before, the stability of the biomarker in the wastewater during the process of sampling and characterization is essential. Furthermore, the biomarker should be unique to the human metabolism of interest to distinguish it from exogenous biomarkers originating from other sources such as microbes or animal contamination in wastewater (Barceló, 2020) . However, it was argued that the genetic materials of SARS-CoV-2 decays naturally below the limits of detection in the environment, including wastewater and aerosol (Hart and Halden, 2020b) , which could affect the quantity and quality of biomarker characteristics in these media.

The selection of appropriate biomarkers can be challenging due to the complexity of the water and wastewater matrix, which requires extensive preparation of samples and pre-concentration of target analytes (Sims and Kasprzyk-Hordern, 2020; Pandopulos et al., 2020) . Wastewater contains complex microbial communities with diverse information on biological and chemical compounds, which are difficult to extract and characterize (Adelodun et al., 2020b) . Similarly, the use of volatile compounds for the biosampling of airborne viruses has so far received less attention due to the challenges in identifying virus-specific volatile compounds (Gould et al., 2020) . Nevertheless, significant progress has been recorded on biomarkers for drug/pharmaceutical monitoring (Daughton, 2020; Sims and Kasprzyk-Hordern, 2020) . SARS-CoV-2 infections are found to cause serious alterations in the respiratory, pulmonary, and gastrointestinal microbiomes (Villapol, 2020; Lamote et al., 2020) , thereby causing changes of metabolites which can be detected in the faeces or breath (Gould et al., 2020) . The viruses contain proteins, RNA, and lipids, which are excreted or expelled via metabolic activity, making them potential marker candidates for viral infection (Gould et al., 2020) . Few studies have also been conducted on the biomarkers for SARS-CoV-2. Zhang and Guo (2020) highlighted three specific biomarker targets for SARS-CoV-2 detection, and these include the viral RNA genome, spike protein, and glycans. Conticini et al. (2020) suggested chemokines and inflammatory cytokines as potential biomarkers for acute respiratory distress syndrome, an elevated condition in COVID-19 resulting from airborne particulates of SARS-CoV-2. Table 3 presents other potential biomarkers proposed in the literature that could be adopted for monitoring SARS-CoV-2 in wastewater and airborne particles using previously identified biomarkers with similar indicators to COVID-19.

J o u r n a l P r e -p r o o f (Syslová et al., 2008; Psathakis et al., 2006) J o u r n a l P r e -p r o o f

The detection of specific pollutants in wastewater related to human lifestyle and health results is an effective non-intrusive, and non-incriminating system for monitoring and returning relevant information for preserving human health and legacy for communities. This can also be an effective way of helping in the early warning of outbreaks as those caused by viral infections. If on one hand

Wastewater based epidemiology resulted already successfully exploited for investigating the use and abuse of both licit and illicit drugs related to lifestyle and health of communities, on the other hand some criticisms remain in its use for detecting viral infections and disease. Adequate concentration and stability of the specific biomarkers in both wastewater and during the sampling and storing procedure resulted in the first critical aspects able to affect the liability of the successive measurements. Another criticism was detected in the absence of standardized and largely agreed methodologies and protocols, in particular for SARS-CoV-2, able to lead to comparable and significant results. Traces of SARS-CoV-2 RNA in the airborne particulate matter were also detected by some studies in the literature, suggesting this medium as another possible way for virus spreading but also monitoring its presence in given areas and communities. By the way, the limited number of studies, the absence of adequate knowledge, and several criticisms concerning the analytical procedures and methodologies, indicates that much more research activity resulted necessarily for better understanding this phenomenon. Better identification of biomarkers with suitable features for viral diseases detection and definition of standardized and adequate procedures for sampling, storage, and analysis of both wastewater and airborne samples are the main aspect requiring further research activity. In view of the above, resulted strongly recommended the encouragement of interdisciplinary, multi-dimensional research activities capable of examining the initial diffusion of the virus as well as its outbreak.

This requires a multiple perspectives approach, including, among the others, biomedical, epidemiological and environmental expertise together with engineering, economic but also political, social and demographic ones.

The authors declare that they have no known potential competing for financial interests or personal relationships that could have appeared to influence the work reported/presented in this review article. All authors have critically read and approved the final version/draft of the manuscript for communication. Faridi, S., Niazi, S., Sadeghi, K., Naddafi, K., Yavarian, J., Shamsipour, M., Jandaghi, N.Z.S., Sadeghiniiat, K., Nabizadeh, R. Lamers, M.M., Beumer, J., van der Vaart, J., Knoops, K., Puschhof, J., Breugem, T.I., Ravelli, R.B., van Schayck, J.P., Mykytyn, A.Z., Duimel, H.Q. and van Donselaar, E., 2020. SARS-CoV-2 productively infects human gut enterocytes. Science. https://doi.org/10.1126/science.abc1669

Snowballing transmission of COVID-19 (SARS-CoV-2) through wastewater: Any sustainable preventive measures to curtail the scourge in low-income countries?

Assessment of socioeconomic inequality based on virus-contaminated water usage in developing countries: A review

First confirmed detection of SARS-CoV-2 in untreated wastewater in Australia: A proof of concept for the wastewater surveillance of COVID-19 in the community

Detection of SARS-CoV-2 RNA in commercial passenger aircraft and cruise ship wastewater: a surveillance tool for assessing the presence of COVID-19 infected travellers

Recognizing and controlling airborne transmission of SARSCoV-2 in indoor environments

Points of Significance: Association, correlation and causation

Coronaviruses in wastewater processes: Source, fate and potential risks

Consideration of the Aerosol Transmission for COVID-19 and Public Health

The coronavirus pandemic and aerosols: Does COVID-19 transmit via expiratory particles?

Diversity, co-occurrence and implications of fungal communities in wastewater treatment plants

Impact of seasonal temperature transition, alkalinity and other abiotic factors on the persistence of viruses in swine and dairy manures

Stigma during the COVID-19 pandemic

Wastewater-Based Epidemiology to Monitor COVID-19 Outbreak: Present and Future Diagnostic Methods to be in Your Radar. Case Stud

An environmental and health perspective for COVID-19 outbreak: Meteorology and air quality influence, sewage epidemiology indicator, hospitals disinfection, drug therapies and recommendations

Regressing SARS-CoV-2sewage measurements onto COVID-19 burden in the population: a proof-of-concept for quantitative environmental surveillance

AIR quality and COVID-19 adverse outcomes: Divergent views and experimental findings

Population Normalization with Ammonium in Wastewater-Based Epidemiology: Application to Illicit Drug Monitoring

Opportunities and Challenges for Biosensors and Nanoscale Analytical Tools for Pandemics: COVID-19

CrAssphage and Pepper Mild Mottle Virus as Viral Water Quality Monitoring Tools-Potential, Research Gaps, and Way Forward

Systematic review and metaanalysis of decay rates of waterborne mammalian viruses and coliphages in surface waters

A review and update on waterborne viral diseases associated with swimming pools

First data analysis about possible COVID-19 virus airborne diffusion due to air particulate matter (PM): The case of Lombardy (Italy)

Commercial exchanges instead of air pollution as possible origin of COVID-19 initial diffusion phase in Italy: more efforts are necessary to address interdisciplinary research

Understanding COVID-19 diffusion requires an interdisciplinary, multi-dimensional approach

Turbulent gas clouds and respiratory pathogen emissions, potential implications for reducing transmission of COVID-19

Evidence-based considerations exploring relations between sars-cov-2 pandemic and air pollution: Involvement of pm2.5-mediated up-regulation of the viral receptor ace

O Impacto do Material Particulado na Qualidade do Ar (Impact of Particulate Matter on Air Quality)

Quantitative assessment of the risk of airborne transmission of SARS-CoV-2 infection: prospective and retrospective applications

RT-qPCR testing of SARS-CoV-2: a primer

Making waves: Coronavirus detection, presence and persistence in the water environment: State of the art and knowledge needs for public health

Survival of surrogate coronaviruses in water

Identification and measurement of illicit drugs and their metabolites in urban wastewater by liquid chromatographytandem mass spectrometry

CDC (Centers of Disease Control and Prevention). 2020. Coronavirus Disease

A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster

Middle East Respiratory syndrome coronavirus: another zoonotic betacoronavirus causing SARS-like disease

Towards finding a population biomarker for wastewater epidemiology studies

COVID-19 disease with positive fecal and negative pharyngeal and sputum viral tests

The presence of SARS-CoV-2 RNA in the feces of COVID-19 patients

The effect of latitude and PM 2.5 on spreading of SARS-CoV-2 in tropical and temperate zone countries

Gastrointestinal manifestations of SARS-CoV-2 infection and virus load in fecal samples from the Hong Kong cohort and systematic review and meta-analysis

Detection of air and surface contamination by SARS-CoV-2 in hospital rooms of infected patients

SARS-CoV-2 concentrations and virus-laden aerosol size distributions in outdoor air in north and south of Italy

Wastewater-based epidemiology biomarkers: Past, present and future

Factors determining the diffusion of COVID-19 and suggested strategy to prevent future accelerated viral infectivity similar to COVID

Air Pollution and Covid-19: The Role of Particulate Matter in the Spread and Increase of Covid-19's Morbidity and Mortality

Can atmospheric pollution be considered a co-factor in extremely high level of SARS-CoV-2 lethality in Northern Italy?

The zoonotic potential of rotavirus

Origin and evolution of pathogenic coronaviruses

Illicit Drugs in Municipal Sewage: Proposed New Non-Intrusive Tool to Heighten Public Awareness of Societal Use of Illicit/Abused Drugs and Their Potential for Ecological Consequences. Pharmaceuticals and Personal Care Products on the Environment: Scientific and Regulatory Issue

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing

Breath analysis for detection of viral infection, the current position of the field

Quantification of SARS-CoV-2 and cross-assembly phage (crAssphage) from wastewater to monitor coronavirus transmission within communities

Clinical characteristics of coronavirus disease 2019 in China

Three emerging coronaviruses in two decades: the story of SARS, MERS, and now COVID-19

Occurrence and fate of antibiotic, analgesic/anti-inflammatory, and antifungal compounds in five wastewater treatment processes

SARS-CoV-2 in river water: Implications in low sanitation countries

Survival of coronaviruses in water and wastewater

Persistent viral shedding of SARS-CoV-2 in faeces -a rapid review. Color. Dis. 611-620

First environmental surveillance for the presence of SARS-CoV-2 RNA in wastewater and river water in Japan

Computational analysis of SARS-CoV-2/COVID-19 surveillance by wastewater-based epidemiology locally and globally: Feasibility, economy, opportunities and challenges

Modeling wastewater temperature and attenuation of sewageborne biomarkers globally

Temporal dynamics in viral shedding and transmissibility of COVID-19

Surveillance of Influenza A and the pandemic influenza A (H1N1) 2009 in sewage and surface water in the Netherlands

Detection of pathogenic viruses in sewage provided early warnings of hepatitis a virus and norovirus outbreaks

Estimating the prevalence of hepatitis B by wastewater-based epidemiology in 19 cities in China

Isoprostanes-biomarkers of lipid peroxidation: Their utility in evaluating oxidative stress and analysis

Asymptomatic SARS-CoV-2 infected case with viral detection positive in stool but negative in nasopharyngeal samples lasts for 42 days

Current trends in Finnish drug abuse: Wastewater based epidemiology combined with other national indicators

The removal of pharmaceuticals, personal care products, endocrine disruptors and illicit drugs during wastewater treatment and its impact on the quality of receiving waters

One-year monthly quantitative survey of noroviruses, enteroviruses, and adenoviruses in wastewater collected from six plants in Japan

The first application of wastewater-based drug epidemiology in five South Korean cities

The scent of COVID-19: Viral (semi-)volatiles as fast diagnostic biomarkers?

Environmental release of oseltamivir from a Norwegian sewage treatment plant during the 2009 influenza A (H1N1) pandemic

The role of wastewater treatment plants as tools for SARS-CoV-2 early detection and removal

Respiratory virus shedding in exhaled breath and efficacy of face mask

Is the coronavirus airborne? Experts can't agree

Monitoring and evaluation of infectious rotaviruses in various wastewater effluents and receiving waters revealed correlation and seasonal pattern of occurrences

Air pollution and temperature are associated with increased COVID-19 incidence: A time series study

Gastrointestinal symptoms of 95 cases with SARS-CoV-2 infection

Aerodynamic analysis of SARS-CoV-2 in two Wuhan hospitals

Evaluation of SARS-CoV-2 RNA shedding in clinical specimens and clinical characteristics of 10 patients with COVID-19 in Macau

SARS-CoV-2 in wastewater: potential health risk, but also data source

Effects of temperature variation and humidity on the death of COVID-19 in Wuhan

Illicit drugs consumption evaluation by wastewater-based epidemiology in the urban area of Palermo city (Italy)

Performance evaluation of human-specific viral markers and application of pepper mild mottle virus and crAssphage to environmental water samples as fecal pollution markers in the Kathmandu Valley

Can a paper-based device trace COVID-19 sources with wastewater-based epidemiology?

Droplet fate in a cough puff

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

Sewage analysis as a tool for the COVID-19 pandemic response and management: the urgent need for optimised protocols for SARS-CoV-2 detection and quantification

Influenza virus aerosols in human exhaled breath: Particle size, culturability, and effect of surgical masks

SARS-CoV-2 in Detroit Wastewater

Assessing the risk of COVID-19 from multiple pathways of exposure to SARS-CoV-2: modeling in health-care settings and effectiveness of nonpharmaceutical interventions

Estimating the asymptomatic proportion of coronavirus disease 2019 (COVID-19) cases on board the Diamond Princess cruise ship

ICTV virus taxonomy profile: polyomaviridae

It is time to address airborne transmission of COVID-19

Airborne transmission of SARS-CoV-2: The world should face the reality

Safe drinking water and waterborne outbreaks

Tracing surface and airborne SARS-CoV-2 RNA inside public buses and subway trains

Editorial Perspectives: 2019 novel coronavirus (SARS-CoV-2): what is its fate in urban water cycle and how can the water research community respond

National Research Council, 2020. Rapid Expert Consultation on the Possibility of Bioaerosol Spread of SARS-CoV-2 for the COVID-19 Pandemic

Temporal Detection and Phylogenetic Assessment of SARS-CoV-2 in Municipal Wastewater

The COVID-19 pandemic: Considerations for the waste and wastewater services sector

Subcellular location and topology of severe acute respiratory syndrome coronavirus envelope protein

A systematic review of possible airborne transmission of the COVID-19 virus (SARS-CoV-2) in the indoor air environment

Prevalence of norovirus and factors influencing virus concentrations during one year in a full-scale wastewater treatment plant

What do we know about the SARS-1 CoV-2 coronavirus in the environment?

Removal of Viruses in Membrane Bioreactors

A water-focused one-health approach for early detection and prevention of viral outbreaks

COVID-19 and Recycled Wastewater Irrigation: A Review of Implications

Air, Surface Environmental, and Personal Protective Equipment Contamination by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) from a Symptomatic Patient

Regressing SARS-CoV-2 sewage measurements onto COVID-19 burden in the population: a proof-of-concept for quantitative environmental surveillance

Prevalence of Asymptomatic SARS-CoV-2 Infection

Clinical characteristics of COVID-19 patients with digestive symptoms in Hubei, China

Application of catecholamine metabolites as endogenous population biomarkers for wastewater-based epidemiology

A sensitive analytical method for the measurement of neurotransmitter metabolites as potential population biomarkers in wastewater

Making waves: Wastewater-based epidemiology for SARS-CoV-2

Developing robust approaches for surveillance and prediction is harder than it looks

Seasonal Influences on the Spread of SARS-CoV-2 (COVID19), Causality, and Forecastabililty (3-15-2020)

Detection of SARS coronavirus in patients with severe acute respiratory syndrome by conventional and real-time quantitative reverse transcription-PCR assays

Antiviral drugs in wastewater and surface waters: a new pharmaceutical class of environmental relevance?

Exhaled markers of oxidative stress in idiopathic pulmonary fibrosis

SARS-CoV-2 RNA in wastewater anticipated COVID-19 occurrence in a low prevalence area

Using biomarkers in wastewater to monitor community drug use: A conceptual approach for dealing with new psychoactive substances

Analysis and interpretation of specific ethanol metabolites, ethyl sulfate, and ethyl glucuronide in sewage effluent for the quantitative measurement of regional alcohol consumption

Estimating population size in wastewater-based epidemiology. Valencia metropolitan area as a case study

Presence and infectivity of SARS-CoV-2 virus in wastewaters and rivers

The occurrence of selected pharmaceuticals in wastewater effluent and surface waters of the lower Tyne catchment

Liquid chromatography-high resolution mass spectrometry with immunoaffinity clean-up for the determination of the oxidative stress biomarker 8-iso-prostaglandin F2alpha in wastewater

Aerosol and surface contamination of SARS-CoV-2 observed in quarantine and isolation care

Occurrence and behavior of macrolide antibiotics in municipal wastewater treatment: possible importance of metabolites, synthesis byproducts, and transformation products

Evaluation of the potential relationship between Particulate Matter (PM) pollution and COVID-19 infection

SARS-Cov-2 RNA Found on Particulate Matter of Bergamo in Northern Italy: First Preliminary Evidence

Application of real-time quantitative PCR for the detection of selected bacterial pathogens during municipal wastewater treatment

First detection of SARS-CoV-2 RNA in wastewater in North America: A study in Louisiana

Assessment of the efficacy of membrane filtration processes to remove human enteric viruses and the suitability of bacteriophages and a plant virus as surrogates for those viruses

Systematic review and meta-analysis of the persistence and disinfection of human coronaviruses and their viral surrogates in water and wastewater

Future perspectives of wastewater-based epidemiology: Monitoring infectious disease spread and resistance to the community level

Detection and survival of SARS-coronavirus in human stool, urine, wastewater and sludge

The M, E and N structural proteins of the SARS coronavirus are required for efficient assembly, trafficking and release of virus-like particles

Prospects of nanomaterialsenabled biosensors for COVID-19 detection

Metagenomic evaluation of the highly abundant human gut bacteriophage CrAssphage for source tracking of human fecal pollution

Co-occurrence of crAssphage with antibiotic resistance genes in an impacted urban watershed

Quantitative CrAssphage PCR assays for human fecal pollution measurement

The airborne lifetime of small speech droplets and their potential importance in SARS-CoV-2 transmission

C-reactive protein is associated with renal function abnormalities in a non-diabetic population

Epidemiology, Genetic Recombination, and Pathogenesis of Coronaviruses

Determination of 8-iso-prostaglandin F2α in exhaled breath condensate using combination of immunoseparation and LC-ESI-MS/MS

Antiviral drugs zanamivir and oseltamivir found in wastewater and surface water in osaka

Aerosol transmission of SARS-CoV-2? Evidence, prevention and control

Post-lockdown detection of SARS-CoV-2 RNA in the wastewater of Montpellier, France. One Health

Existence of SARS-CoV-2 in wastewater: implications for its environmental transmission in developing communities

Aerosol and Surface Stability of SARS-CoV-2 as Compared with SARS-CoV-1

Gastrointestinal symptoms associated with COVID-19: impact on the gut microbiome

Excretion and detection of SARS coronavirus and its nucleic acid from digestive system

The establishment of reference sequence for SARS-CoV-2 and variation analysis

Coronavirus disease 19 infection does not result in acute kidney injury: an analysis of 116 hospitalized patients from Wuhan, China

Limits of detection of 6 approved RT-PCR kits for the novel SARS-Coronavirus-2 (SARS-CoV-2)

Asymptomatic cases with SARS-CoV-2 infection

Coronavirus Pathogenesis and the Emerging Pathogen Severe Acute Respiratory Syndrome Coronavirus/Susan R. Weiss, Sonia Navas-Martin

Detection of SARS-CoV-2 in raw and treated wastewater in Germany-suitability for COVID-19 surveillance and potential transmission risks

Guidelines for Drinking-Water Quality

COVID-19 2020 Situation summary -Updated

Modes of transmission of virus causing COVID-19: implications for IPC precaution recommendations

Virological assessment of hospitalized patients with COVID-2019

Preventing diarrhoea through better water, sanitation and hygiene: exposures and impacts in low-and middle-income countries. World Health Organization

SARS-CoV-2 titers in wastewater are higher than expected from clinically confirmed cases

Prolonged presence of SARS-CoV-2 viral RNA in faecal samples

Time course quantitative detection of SARS-CoV-2 in Parisian wastewaters correlates with COVID-19 confirmed cases

Infectious SARS-CoV-2 in feces of patient with severe COVID-19

Evidence for gastrointestinal infection of SARS-CoV-2

Systematic Comparison of Two Animal-to-Human Transmitted Human Coronaviruses: SARS-CoV-2 and SARS-CoV

Characteristics of pediatric SARS-CoV-2 infection and potential evidence for persistent fecal viral shedding

Infectious virus in exhaled breath of symptomatic seasonal influenza cases from a college community

Survivability, partitioning, and recovery of enveloped viruses in untreated municipal wastewater

An efficient virus aerosol sampler enabled by adiabatic expansion

TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes

Biomarkers of COVID-19 and technologies to combat SARS-CoV-2

Virus Shedding Patterns in Nasopharyngeal and Fecal Specimens of COVID-19 Patients

Airborne transmission may have played a role in the spread of 2015 highly pathogenic avian influenza outbreaks in the United States

Association between ambient temperature and COVID-19 infection in 122 cities from China

China Novel Coronavirus Investigating and Research Team. A novel coronavirus from patients with pneumonia in China

Association between short-term exposure to air pollution and COVID-19 infection: Evidence from China

Use of ceramic membranes in a membrane filtration supported by coagulation for the treatment of dairy wastewater

We thank the Department of Biotechnology and Ministry of Human Resource and Development, Govt. of India for the fellowship to Omkar Indari and Shweta Jakhmola, respectively, in the form of a research stipend. We gratefully acknowledge the Indian Institute of Technology Indore for the facilities and support.J o u r n a l P r e -p r o o f > Wastewater and airborn PM analysis for SARS-CoV-2 surveillance was investigated > RNA of SARS-CoV-2 was detected in several untreated wastewaters > Only correlation but no causal nexus between PM and COVID-19 spreading was detected > More research needed for biomarkes and methodologies definition > Multidisciplinary approach resulted strongly recommended J o u r n a l P r e -p r o o f

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: J o u r n a l P r e -p r o o f