A review of the presence of SARS-CoV-2 RNA in wastewater and airborne particulates and its use for virus spreading surveillance

3.1. Coronaviruses in wastewater: Sources and occurrence and potential risk of infection

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 and Fratini, 2012; Moreira and Bondelind, 2017; Girones, 2017). SARS-CoV-2 is an enveloped virus belonging to the Betacoronavirus genus in the Coronaviride family characterized by positive-sense 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; Ahmed et al., 2020a, Ahmed et al., 2020b). 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 (Xiao et al., 2020b). 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 Table 1 the percentage of patients affected by SARS-CoV-2 representing gastrointestinal symptoms resulted quite high, in general, > 10% up to 62.5%, as the number of patients presenting SARS-CoV-2 RNA fragments in the stools. Furthermore, for those cases investigated, the SARS-CoV-2 load in the samples of stools analyzed resulted in general > 2 E³ (copies/ml).

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) in monitoring one of the major wastewater treatment plants in Massachusetts. Ahmed et al. (2020a) monitored one pumping station and two wastewater treatment plants in Southeast Queensland (Australia), reporting 2 out of 9 samples with the presence of fragments of SARS-CoV-2 RNA. Similar studies were also performed by Wurtzer et al. (2020) and La Rosa et al. (2020) for France and Italy, respectively. All these findings suggested that wastewater could be used for COVID-19 surveillance, but, in any case, no evidence of SARS-CoV-2 infection spreading via water and wastewater was currently detected. Wang et al. (2005) reported a persistence of SARS-CoV in hospital wastewater, domestic sewage, and tap water for 2 days at 20 °C and 14 days at 4 °C. The same authors also reported that at 20 °C, the persistence of the virus in faeces and urine was of 3 days and 17 days, respectively, whereas a concentration of 10 mg/L of chlorine with a contact time ≥10 min resulted able to completely inactivate SARS-CoV. For a chlorine concentration of 20 mg/L the minimum contact time decreased to 1 min. Concerning the survival of coronavirus in tap and wastewater, Ahmed et al., 2020a, Ahmed et al., 2020b compared HCoV-229E and animal FIPV coronavirus with Poliovirus-1 (PV-1). In wastewater at 23 °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, Wu et al., 2020b, Wu et al., 2020a 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. (2020a) proposed two methods: a first method based on direct RNA extraction from the electronegative 0.45 -μm filter 90 mm diameter; a second method based on centrifugation at 4750 rpm for 30 min followed by centrifugation of the supernatant at 3500 rpm for 15 min through a centrifugal filter with a cut of 10 kDa. Wurtzer et al. (2020) proposed ultracentrifugation at 200,000 rpm for 1h at 4 °C. For RNA detection, La Rosa et al. (2020) used three different PRC assays: a broad range of PCR for Coronavirus detection targeting ORF1ab; a nested RT-PCR targeting the spike regions for SARS-CoV-2 proposed by 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.

3.2. SARS-CoV-2 in airborne particulate matter

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 virus, including the role of particulate matter, are not clear. In this context, the number of studies concerning the role of PM₁₀/PM_2.5 as carriers of virus spreading is rapidly growing. Airborne particles containing the virus can be emitted by infected individuals during sneeze, cough, shouting, speaking and normally breathing as already reported for other respiratory viruses belonging or not to the Coronavirinae subfamily (Milton et al., 2013; Wu et al., 2020b, Wu et al., 2020a; Leung et al., 2020). Settling rapidity of larger size respiratory droplets (i.e. > 5 μm) 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 (Wu et al., 2020b, Wu et al., 2020a). 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 10⁵-10⁶) 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 (Barceló, 2020b). With specific reference to the role of PM₁₀/PM_2.5 as potentially 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 (Wu et al., 2020b, Wu et al., 2020a; 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 cause-effect 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 et al., 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, Wu et al., 2020b, Wu et al., 2020a highlighted how the concentration of PM2.5 can affect the distance in which the virus can be transmitted because SARS-CoV-2 droplets absorb PM2.5 and form a solid/liquid (PM2.5/SARS-CoV-2) interface. Zhao et al. (2019) found a similar result through a modelling analysis applied to the virus of Pathogenic Avian Influenza (HPAI). They stated that PM2.5 might transport the virus for longer distances than PM10, due to the lower deposition velocity. However, higher distances contribute to a lower probability of infection because of the decrease of the concentration of virus/particles. Concerning the RNA analysis approach, only the study of Setti et al. (2020b) was identified. Here, 34 PM₁₀ samples were collected from an industrial site in the province of Bergamo in Northern Italy, over three weeks (from February 21st to March 13th, 2020) and in 7 samples have identified the presence of SARS-COV-2 RNA. About this approach, some general considerations should be reported:

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  The extraction method has not yet been well standardized;

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  The number of samples is statistically low to deduct general considerations and further studies are necessary;

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  The evidence of viral RNA is not indicative of the presence of active viruses that could be effectively transmitted.

In conclusion, based on what emerged from the analysis of the scientific literature, there is no scientific evidence of a possible spread of COVID-19 infection through atmospheric particulate matter. Furthermore, these findings resulted characterized by a high level of uncertainty preventing any definitive scientifically valid conclusions.

4.1. Protocols for WBE

The practical exploitation of WBE requires the definition of clear and agreed methodologies including all different phases from wastewater sampling to the final correlation of biomarker concentration and health status. Based on the current aim of this analysis (i.e., lifestyle or disease surveillance) and on the biomarkers used for this goal, the methodology proposed in the literature showed some differences. In any case, a common preliminary step of all the methodologies consists in the study of the area and of the population served by the sewage system for the exact identification of the size of the population interested and the presence of other wastewaters different from the urban ones entering in the sewage system (McCall et al., 2020).

4.2. SARS-CoV-2 detection

In general, as reported by several authors (Ahmed et al., 2020a; Hart and Halden, 2020a, Hart and Halden, 2020b; 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; Wu 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., 2019; 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; Wu 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 (PMMoV), adenovirus, human polyomavirus, torque teno virus, and norovirus, CrAssphage in the sewage of the United States and European Union countries. Many of the researchers were found CrAssphage appears to be human-associated; however, it has been detected in a limited number of seagulls, dog, duck, pig, and chicken faecal samples (Malla et al., 2019; Stachler et al., 2017). Countries like United States, United Kingdom, Australia, Japan, Thailand, and Nepal were successfully deployed CrAssphage to detect faecal pollution in environmental waters (Stachler et al., 2019; Farkas et al., 2020; Kongprajug et al., 2019; Bivins et al., 2020).

A recent study by Haramoto et al. (2020) found detection of SARS-CoV-2 RNA in wastewater through qPCR technique and none of the samples of river water was found positive for SARS-CoV-2 RNA. They found virus detection in the secondary treated wastewater was 1.4 × 10²–2.5 × 10³ copies/L. The survival and stability of viruses in wastewater depend on physicochemical conditions such as temperature, pH, solids, and the presence of micropollutants (Fongaro et al., 2019). Auffret et al. (2019) showed that the detrimental effect on virus persistence with high alkalinity levels of wastewater. Bivins et al. (2020) reported a 90% reduction of viable SARS-CoV-2 in wastewater and tap water through RT-qPCR detection technique at room temperature. Other researchers have reported persistence of viruses at different temperatures. For example Casanova et al. (2009) found a 99% reduction of TGEV and MHV at the temperature of 25 °C in the sewage. Ye et al. (2016) reported a 90% reduction of MHV in unpasteurized wastewater in 13 h at 25 °C. Gundy et al. (2009) found to be 99.9% reduction of feline infectious peritonitis virus and human coronavirus 229E in the primary effluent at 23 °C in 2 and 4 days, respectively. In the case of wastewater from biological treatment processes, the survival and persistence of SARS-CoV-2 RNA are unclear due to insufficient experimental data (Oliver et al., 2020). Guerrero-Latorre et al. (2020) found SARS-CoV-2 RNA in the river samples at concentrations of 2.07 × 10⁵ to 3.19 × 10⁶ gene copies/L. Ahmed et al. (2020) reported that among five different RT-qPCR assays (CDCN1, CDC N2, N_Sarbeco, NIID_2019- nCoV_N, and E_Sarbeco), the CDC N1 assay is the most sensitive one while the N_Sarbeco showed the least sensitivity, while positive samples among wastewater samples of a cruise ship and three aircraft were near the assay LOD (37–40 cycles), providing inconsistent results among the tested assays.

Wastewater and aerosols are ideal surveillance materials for assessing qualitative and quantitative molecular epidemiological information on population exposure to SARS-CoV-2, including biological and chemical markers associated with human activities within a community or a catchment (Carducci et al., 2020; Polo et al., 2020). Chirizzi et al. (2021) measured the concentrations of SARS-CoV-2 distribution in outdoor air samples from North and South of Italy. 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. Fig. 3 depict the bioaerosols sampling and WBE surveillance concept for viral particles in outdoor particulate matter, indoor aerosol, and wastewater treatment plants. One of the challenges of virus identification, unlike other microorganisms, is the inability to mutate outside the living host cells (Polo et al., 2020). The detection of human viruses in the environment can only be facilitated when excreted through faeces and urine or passed out through breath and cough or sneeze with significant concentrations and a half-life long enough to identify and characterize them.

Some studies have also demonstrated the presence of SARS-CoV-2 RNA in aerosol samples. Chia et al. (2020) identified positive SARS-CoV-2 through the environmental sampling of hospital rooms. Santarpia et al. (2020) confirmed the presence of SARS-CoV-2 in the air samples from the hospital and isolation rooms. Liu et al. (2020) detected higher concentrations of SARS-CoV-2 RNA in the aerosol samples from the toilets used by the patients compared to the isolation room. Ong et al. (2020) detected small virus-laden droplets at the air exhaust outlet from the swab samples. Srivastava et al. (2021) also reported the prospect of rapid detection of SARS-CoV-2 in aerosol using nanomaterials enabled biosensing technique. The detection of the genetic material of SARS-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 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; Liu 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, 2020a, Hart and Halden, 2020b). 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.