Several studies have detected SARS-CoV-2 in the stool of infected people as in urban wastewater. The quantification of SARS-CoV-2 in wastewater appears today as a powerful tool that can help in wastewater-based epidemiology (WBE). The goal is to improve the prediction of new waves of COVID-19 outbreaks and provide an early warning of the evolution of the infection. In this research, we highlighted some practical and scientific aspects that emerged during an extensive ongoing monitoring campaign carried out on a large number of wastewater treatment plants located in the province of Trento (North Italy) and aimed at the detection of SARS-CoV-2 in raw municipal wastewater. The open issues underline are related to the collection and storage (sampling protocol, storage and heat treatment), to the molecular analysis (enrichment phase), and to the mathematical calculation of SARS-CoV-2 load in wastewater, suitable for WBE (standard curve to obtain the concentration of genomic units and flow rate measurements). This study provides some insights that can help in the implementation of surveillance plans in other regions.

  • Quantification of SARS-CoV-2.

  • Heat treatment.

  • Storage protocols.

  • Wastewater-based epidemiology.

Since November 2019, the world population has been affected by a pandemic caused by a new virus, the SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) (Coronaviridae Study Group of the International Committee on Taxonomy of Viruses 2020). This virus is a member of the subfamily Coronavirinae, genus Betacoronavirus, subgenus Sarbecovirus (Coronaviridae Study Group of the International Committee on Taxonomy of Viruses 2020) to which also belong the viruses SARS and MERS, which have caused major outbreaks in recent decades. The disease caused by this virus, called COVID-19, mainly affects the respiratory system causing fever, cough, rhinorrhea, dyspnoea, or severe pneumonia (Chen et al. 2020; Guan et al. 2020). An important interaction is with the gastrointestinal system, which may lead to diarrhoea, vomiting, or abdominal pain (Guan et al. 2020; Pan et al. 2020) that may be early symptoms of the disease (Huang et al. 2020; Lai et al. 2020). SARS-CoV-2 can be detected in the stool of positive patients (Wang et al. 2020), even if asymptomatic (Park et al. 2020), with titres in the range of 103–107 genomic unit/g faeces (GU/g) depending on the day of sampling (Wölfel et al. 2020). The presence of SARS-CoV-2 in faeces leads to finding it in the urban sewers and wastewater treatment plants (WWTPs). When faeces reach the sewerage, the viral titre undergoes a dilution of about 103 times due to the discharge of drinking water, rainwater, or infiltrations, and the concentration of SARS-CoV-2 drops to 1–103 GU/mL (Foladori et al. 2020). The quantification of SARS-CoV-2 in wastewater could give additional information about the spread of the COVID-19 outbreak (Daughton 2020).

This is the topic of the field of wastewater-based epidemiology (WBE), which deals with the use of quantitative measurement of human biomarkers in sewage to evaluate lifestyle, health, and exposure at the community level (https://score-cost.eu/; Sims & Kasprzyk-Hordern 2020). Several nations have moved to develop a monitoring network for their wastewater. For example, in Italy, the severe acute respiratory syndrome (SARI) project was born for the surveillance of SARS-CoV-2 at national level. However, there is still a lack of agreement on an international standard protocol. For monitoring a large number of WWTPs in a territory, sampling, storage, and analysis of samples should be fast and affordable, with moderate efforts in terms of personnel, equipment, and costs, but at the same time ensuring reliable results. The choice of the method is today something closely linked to the skills and availability of each workgroup. For these reasons, the quantification of SARS-CoV-2 in wastewater for WBE applications remains an open issue (Table 1).

In this research we highlighted some practical and scientific aspects that emerged during an extensive ongoing monitoring campaign in August 2020 to January 2021, aimed at quantifying SARS-CoV-2 in 31 WWTPs in the Trento province (North Italy). The monitored WWTPs had daily average flow rates range from 4,000 m3/d to 40,000 m3/d and a treatment capacity range from 6,000 to 85,000 residents served. This study provides various insights that can be exploited in the implementation of surveillance plans in other regions.

In the quantification of SARS-CoV-2 in raw wastewater the main areas worthy of further thought are the following: (i) collection and storage of samples, (ii) some steps of the molecular analysis, (iii) mathematical calculation of the viral load in wastewater, suitable for WBE.

Issues related to the collection and storage of wastewater samples

Sampling protocol

The type of sampling may affect the accurate estimation of SARS-CoV-2 content in wastewater, especially for WBE. In general, composite samples are recommended (EPA 2013). They can be formed from: (1) aliquots of wastewater with equal volume taken at equal interval times throughout the day, or (2) aliquots of wastewater with volume proportional to the flow rate, which is often impracticable because this requires a flow measurement device not always available in WWTPs.

To evaluate the fluctuations of SARS-CoV-2 in influent wastewater over the day (24 h), 12 samples collected every 2 hours for 24 hours with an automatic sampler, were analysed. Concentrations, expressed as viral genomic units per litre (GU/L), are shown in Figure 1. Concentration was lower at night while it was higher in the morning, between 9 and 11, due to the human behaviour patterns, e.g. toilet usage, in the early hours, and the corrivation time in the sewerage that causes a delay of approximately 2 hours. Another peak occurred in the afternoon at 15–17. The average concentration was 1.4E + 0.4 GU/L, while the minimum-maximum range was 8.8E + 03–2.9E + 04 GU/L.

Figure 1

Profile of SARS-CoV-2 concentration in 2-h composite samples over the day.

Figure 1

Profile of SARS-CoV-2 concentration in 2-h composite samples over the day.

Due to these fluctuations, the choice of composite samples is preferable for the WBE (Ahmed et al. 2020b). However, grab samples can instead be used to evaluate the peaks of SARS-CoV-2 entering the WWTP (Ahmed et al. 2020b), to evaluate its treatment capacity.

Sample storage

Samples should be maintained at low temperature (4 °C) during collection and transport to the laboratory to preserve the viral load and viability (Medema et al. 2020). With regards to sample storage, it is worth noting that freeze–thaw cycles can damage the integrity of viral RNA. The optimal temperature and duration of the storage are key factors, but at the moment there is not full international agreement on these aspects. Storage at −80 °C, although recommended by some authors (Medema et al. 2020), is not feasible in an extensive monitoring plan with many WWTPs, due to the need to store tens of litres per week. Conversely, the storage at 4 °C or −20 °C appears more feasible. In order to investigate the influence of these two storing temperatures in our monitoring, various aliquots of the same wastewater sample were stored at 4 °C and −20 °C and analysed at 0, 7, 10, 14 and 31 days. As can be seen from the trends in Figure 2, a marked loss of copies occurred in the samples stored at −20 °C; this loss happened especially in the first 10 days of storage, with a reduction of 0.5 log. Conversely, the number of copies did not decrease significantly when stored at 4 °C for 14 days. This is in agreement with the findings of Medema et al. (2020), who indicated that storage at 4 °C is preferable as the viral RNA remains intact for the first 15 days (Medema et al. 2020). By contrast, Xiao et al. (2020) indicated to keep the samples at 4 °C when the analysis is performed in the following 24 h, otherwise for longer storage (>24 h) the samples should be kept at −20 °C. Therefore, the influence of 4 °C for longer times requires further investigation, especially to find an explanation to the increase phenomena observed.

Figure 2

Trends of SARS-CoV-2 concentration during storage at 4 °C and −20 °C over time.

Figure 2

Trends of SARS-CoV-2 concentration during storage at 4 °C and −20 °C over time.

Pre-treatment

The thermal pre-treatment, aimed at inactivating the virus and making the sample safer for the operators, is another aspect on which a shared agreement has not been reached yet. Some protocols propose pasteurization at 56 °C for 50 min (La Rosa et al. 2020), but others advise against it, to avoid a loss of viral RNA integrity, so scarce in wastewater (Weidhaas et al. 2020). To investigate the influence of heat treatment on viral RNA detection, four samples from four WWTPs were analysed with and without pasteurization (thermal bath, 56 °C, 30 min). The results are compared in Table 2 in terms of Ct and GU/L. The Ct values in the heat-treated samples were always lower than in the not heat-treated ones. Therefore, a higher concentration of the virus was estimated in the inactivated samples. The heat treatment makes viral RNA more easily detectable. After statistical analysis with the two-way ANOVA test with randomized blocks, the p was significantly lower than the critical p, underlining the important effect of the heat treatment (p = 0.001) in our experimental results. The enrichment phase of the analysis protocol used for the pasteurization test was different from that used for the sampling test and for the storage test. The use of these different methods led to a difference in results of about two orders of magnitude. The comparison between these two protocols will be addressed in the following paragraph.

Table 1

Available methods in the literature for the detection of SARS-CoV-2 in wastewater

Ref.SamplingStorageHeat treat.Enrichment concentrationExtraction
Ahmed et al. (2020a)  Automated and grab sampling techniques Samples were transported on ice to the laboratory and stored at 4 °C  Direct RNA extraction from electronegative membranes or ultrafiltration For RNA extraction, two kits (RNeasy PowerWater Kit and RNeasy PowerMicrobiome Kit; Qiagen, Germany) were used 
La Rosa et al. (2020)  24-hour composites collected from the WWTP influent Immediately stored at −20 °C 30 min treat. at 57 °C Two-phase (PEG–dextran method) separation The viral nucleic acids extraction foresaw the use of the NucliSENS miniMAG semi-automated extraction system with magnetic silica carried out following the manufacturer's instructions (Biomerieux, France).
Before molecular tests, the extracted nucleic acids were further purified from PCR inhibitors 
Medema et al. (2020)  24 h flow-dependent composite sample Samples were transported on ice to the laboratory and stored at 4 °C  The sample was filtered through a Centricon® Plus-70 centrifugal ultrafilter with a cut-off of 10 kDa (Millipore, Amsterdam, the Netherlands) Method 1: RNeasy PowerMicrobiome Kit (Qiagen, Germany).
Method 2: magnetic extraction reagents of the NucliSENS kit (Biomerieux, the Netherlands) in combination with the semi-automated KingFisher mL (Thermo Scientific, The Netherlands) purification system to extract RNA 
Randazzo et al. (2020)   Collected samples were transferred on ice to the laboratory  Aluminium hydroxide adsorption–precipitation concentration method Viral RNA was extracted from concentrates using the NucleoSpin RNA virus kit (Macherey-Nagel GmbH & Co., Germany) 
Wu et al. (2020)  24-h composite samples of raw water  Pasteuriz. at 60 °C for 90 min to inactivate the virus Concentration with polyethylene glycol 8000 (8% w/v, Millipore Sigma) and centrifugation  
Ref.SamplingStorageHeat treat.Enrichment concentrationExtraction
Ahmed et al. (2020a)  Automated and grab sampling techniques Samples were transported on ice to the laboratory and stored at 4 °C  Direct RNA extraction from electronegative membranes or ultrafiltration For RNA extraction, two kits (RNeasy PowerWater Kit and RNeasy PowerMicrobiome Kit; Qiagen, Germany) were used 
La Rosa et al. (2020)  24-hour composites collected from the WWTP influent Immediately stored at −20 °C 30 min treat. at 57 °C Two-phase (PEG–dextran method) separation The viral nucleic acids extraction foresaw the use of the NucliSENS miniMAG semi-automated extraction system with magnetic silica carried out following the manufacturer's instructions (Biomerieux, France).
Before molecular tests, the extracted nucleic acids were further purified from PCR inhibitors 
Medema et al. (2020)  24 h flow-dependent composite sample Samples were transported on ice to the laboratory and stored at 4 °C  The sample was filtered through a Centricon® Plus-70 centrifugal ultrafilter with a cut-off of 10 kDa (Millipore, Amsterdam, the Netherlands) Method 1: RNeasy PowerMicrobiome Kit (Qiagen, Germany).
Method 2: magnetic extraction reagents of the NucliSENS kit (Biomerieux, the Netherlands) in combination with the semi-automated KingFisher mL (Thermo Scientific, The Netherlands) purification system to extract RNA 
Randazzo et al. (2020)   Collected samples were transferred on ice to the laboratory  Aluminium hydroxide adsorption–precipitation concentration method Viral RNA was extracted from concentrates using the NucleoSpin RNA virus kit (Macherey-Nagel GmbH & Co., Germany) 
Wu et al. (2020)  24-h composite samples of raw water  Pasteuriz. at 60 °C for 90 min to inactivate the virus Concentration with polyethylene glycol 8000 (8% w/v, Millipore Sigma) and centrifugation  
Table 2

Comparison of Ct and SARS-CoV-2 concentrations in four different wastewater analysed with and without heat treatment at 56 °C for 30 min

WWTP 1
WWTP 2
WWTP 3
WWTP 4
No heat treatmentHeat treatmentNo heat treatmentHeat treatmentNo heat treatmentHeat treatmentNo heat treatmentHeat treatment
Ct 35.96 33.11 36.59 33.44 35.85 32.31 35.6 33.03 
GU/L 994 3,201 393 1,829 814 6,952 630 4,105 
WWTP 1
WWTP 2
WWTP 3
WWTP 4
No heat treatmentHeat treatmentNo heat treatmentHeat treatmentNo heat treatmentHeat treatmentNo heat treatmentHeat treatment
Ct 35.96 33.11 36.59 33.44 35.85 32.31 35.6 33.03 
GU/L 994 3,201 393 1,829 814 6,952 630 4,105 

Issues related to the molecular analysis

The molecular analysis of the SARS-CoV-2 virus in wastewater is based on three main steps: (1) enrichment, (2) extraction, and (3) amplification.

Comparison between two methods for enrichment

The very low titre of SARS-CoV-2 in wastewater and the need to recover the total amount of the virus for WBE applications, make the enrichment step very critical. In this research the concentration of SARS-CoV-2 in the wastewater from 31 WWTPs was 3–600 GU/mL. Without an efficient enrichment virus detection in wastewater is not currently possible. In this research two enrichment methods were compared (Figure 3): (i) biphasic system (PEG–dextran) according to La Rosa et al. (2020) and adapted from the WHO procedure for Poliovirus; (ii) centrifugation at 12,000 g with the addition of PEG, according to Wu et al. (2020) except for the first step of 0.2 μm filtration. These methods differ for the time of application: (i) overnight for the biphasic system; (ii) only 2 h for centrifugation. Another difference is the role of solids. A complete elimination of solids is indeed required in the centrifugation method (Wu et al. 2020), while in the biphasic method the solids and supernatant are first separated and then combined after the PEG–dextran precipitation (La Rosa et al. 2020), according to the scheme in Figure 3. To specifically test for the difference in the two enrichment methods, the same protocol of viral extraction and RT-PCR was used to obtain Ct values.

Figure 3

Scheme of the analytical approach for the detection of SARS-CoV-2 with the two enrichment methods compared in this study. Please refer to the online version of this paper to see this figure in colour: http://dx.doi.org/10.2166/wst.2021.469.

Figure 3

Scheme of the analytical approach for the detection of SARS-CoV-2 with the two enrichment methods compared in this study. Please refer to the online version of this paper to see this figure in colour: http://dx.doi.org/10.2166/wst.2021.469.

Analysing samples of different WWTPs with both enrichment methods, a significant difference of results was obtained: Ct were in the range 32.73–38 for the biphasic system and 26.73–36.33 for the centrifugation system. This difference is probably due to the PCR-inhibitory substances (e.g., like humic, and fulvic acids) contained in solids that can prevent the correct amplification of viral RNA, producing false-negative results. The presence of inhibitory substances is another critical factor, beyond the low concentration and the poor integrity of the nucleic acids, in the detection of SARS-CoV-2 RNA in wastewater. This set of limitations is the reason why, unlike for respiratory tract specimens, there is still no standard method for the detection of SARS-CoV-2 in wastewater.

Extraction and amplification

For the extraction and amplification steps, different solutions and combinations exist but there is no evidence of a greater efficacy of one protocol over another. Viral RNA extraction is mainly performed with commercially available kits and serves to isolate RNA and clean it of PCR pollutants and inhibitors (Ahmed et al. 2020a; La Rosa et al. 2020; Randazzo et al. 2020). In this research, the extraction phase consists in (Figure 3): (i) lysis of the virus, (ii) bond between RNA and magnetic silica beads, (iii) washing of impurities with buffers, (iv) final elution. Then the pure RNA is used in the amplification step performed through RT-PCR, also called quantitative PCR (qPCR), and based on the exponential amplification of specific genomic regions of the virus (ORF1ab region in our research). The results of RT-PCR are expressed as Ct (threshold cycle) that is the number of cycles required for the fluorescent signal to cross a threshold. Ct is inversely related to the amount of virus. For example, a low Ct value means that a lower number of amplification cycles are required to surpass the threshold and thus the viral concentration in the sample is higher.

Issues related to the mathematical calculation of SARS-CoV-2 load for WBE

Calculation of GU/L using standard curves

The concentration of SARS-CoV-2 RNA in a sample, expressed as GU/L, can be calculated from the threshold cycle (Ct) values and using a standard curve, obtained with a series of dilutions of a standard with known amounts of viral RNA (e.g., AccuPlex SARS-CoV-2 Reference Material Kit used in this study). Figure 4 shows some daily PCR standard curves and their average curve: the slope measures the efficiency of the reaction (maximum efficiency corresponds to a slope of –3.32), the correlation coefficient (R2) measures the fitting of the data with the curve and the Y-intercept is the theoretical limit of detection of the reaction. Once the Ct of the sample is known, the concentration of SARS-CoV-2 can be easily calculated. Our extensive monitoring campaign has highlighted the need to run a standard curve on each RT-PCR run in which the samples of interest are analysed, in order to obtain comparable results, because of the efficiency variability between the different runs of qPCR.

Figure 4

Examples of daily standard curves for the conversion of Ct into GU/L and mean curve. Please refer to the online version of this paper to see this figure in colour: http://dx.doi.org/10.2166/wst.2021.469.

Figure 4

Examples of daily standard curves for the conversion of Ct into GU/L and mean curve. Please refer to the online version of this paper to see this figure in colour: http://dx.doi.org/10.2166/wst.2021.469.

Calculation of the SARS-CoV-2 load in wastewater using influent flow rate

Finally, the viral load of SARS-CoV-2 in the influent wastewater can be calculated by multiplying the value of GU/L by the flow rate entering the WWTP during the time interval of sampling. It is worth noting that the real influent flow rate cannot be estimated using the theoretical calculation that is based on the average daily water consumption per capita (L PE−1d−1) and the population equivalent (PE), because the real flow rate is almost always affected markedly by industries, stormwater, infiltrations or leakages.

This monitoring campaign was carried out on 31 WWTPs with more than 100 samples was a robust basis with which to highlight some practical and scientific aspects and advice, such as the use of heat treatment for a better detection of viral RNA, the feasibility of centrifugation to fasten the enrichment or the use of composite samples for WBE. Other aspects still need to be confirmed, such as the influence of different temperatures for the long-term storage of samples, or the role of inhibitory substances present in the wastewater in PCR analysis.

This research was supported by the Internal Call 2020 ‘Covid 19’, project ‘Surveillance of COVID-19 Pandemic with a Wastewater-Based Epidemiology approach (SCOPE)’, awarded by the University of Trento, Italy. The authors also acknowledge funding from the Italian Ministry of Education, University and Research (MIUR) in the frame of the ‘Departments of Excellence’ grant L. 232/2016.

All relevant data are included in the paper or its Supplementary Information.

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