Feces are the main contribution of SARS-CoV-2 presence in sewage, thus selected target genes were likewise detected by several research groups in feces or from rectal swabs 14,17,22. Detection of several genes is vital as the RNA virus in wastewater is exposed to many environmental factors. Therefore, RNA may be strongly disintegrated resulting in poor amplification and variable detection of individual viral particles. We focused on detecting four genes ORF1ab, S, E and RdRp gene capturing different regions of the virus 23. Similar target genes (S, RdRP, ORF1ab) were also selected by La Rossa et al. 24. On the contrary, other researchers were investigating the presence of 3 different regions of nucleocapsid (N) gene in wastewater 25. Currently, there is no consensus of targets recommended for detecting SARS-CoV-2 in sewage.
In our study 29 out of 52 analyzed wastewater samples (56%) were found to be positive. Table 2 displays CT values of detected genes in both WWTPs at given dates. First positive results were obtained on 29th September with detected S gene at both WWTPs and ORF1ab gene at WWTP Bratislava-Petrzalka. In Bratislava - Centrum lowest CT detected was 31.8, whereas in Bratislava - Petrzalka, it was 32.7 on the same date. At least one of the targets was detected since this date at chosen WWTPs. The most often detected target was the ORF1ab gene with positivity of 33% (22 out of 66) from all positively detected targets.
According to calibration curves, CTs presented in Table 2 were recalculated to the number of viral particles per milliliter. To calculate the daily loads of SARS-CoV-2 in wastewater, viral particles per ml were then multiplied by daily influents measured at the WWTPs (Supplementary Table S2). Then reported numbers of positive RT-qPCR tests and reported numbers of death cases were compared to the number of viral particles in wastewater in Bratislava (Fig. 2).
By interpolating the measured viral particles in the wastewater, we obtained a model useful for comparing the time series of positive RT-qPCR tests, death cases and viral particles (Fig. 2). Since the graph reveals apparent time shifts, we focused on the time lags between particular time series in our analysis.
The following Table 3 describes the dependencies between the time lags of reported positive RT-qPCR tests or death cases and the number of viral particles found on individual days in the wastewater of Bratislava.
It can be seen that the values of R2 for dependence between viral particles in wastewater and reported positive RT-qPCR tests and death cases (Table 3, Supplementary Fig. S2, S3) are better for the weekly time series than for the daily time series. This led us to focus our analysis on weekly time series. This decision is supported by the results of the ANOVA test where the significant difference among the means of the number of positive RT-qPCR tests in the particular days of the week was confirmed (p-value=0.0054, F-value=3.178, DF=6, Supplementary Fig. S1). Therefore, we decided to use the time series of cumulative data with a 7 days period. The weekly time series of measured viral particles in wastewater reported numbers of positive RT-qPCR tests and reported numbers of death cases without/with the appropriate time lags are depicted in supplementary Fig. S4.
By analyzing the dependence between the weekly time series of measured viral particles in the wastewater and reported numbers of positive RT-qPCR tests with a time lag of 2 weeks, the models for estimation of the number of positive RT-qPCR tests were obtained. The results of fitting a linear (R2=81.72%, p-value<0.0001, F-value=116.2, DF=26, Supplementary Fig. S5) and double square root (R2=83.78%, p-value<0.0001, F-value=134.3, DF=26, Supplementary Fig. S6) models describe the relationship between the weekly time series of measured viral particles in the wastewater and reported numbers of positive RT-qPCR tests with the time lag of 2 weeks. The equations of the fitted models are:
Double Square Root:
The models allow estimating the number of positive RT-qPCR tests with model fitting more than 80% (Figure S7).
By analyzing the dependence between the weekly time series of measured viral particles in the wastewater and reported numbers of death cases with a time lag of 4 weeks, the models for estimation of the number of death cases were also obtained. The results of fitting a linear (R2=48.06%, p-value<0.0001, F-value=24.06, DF=26, Supplementary Fig. S8) and square root-Y logarithmic-X (R2=83.21%, p-value<0.0001, F-value=128.83, DF=26, Supplementary Fig. S9) models describe the relationship between the weekly time series of measured viral particles in the wastewater and reported numbers of death cases with the time lag of 4 weeks. The values of R2 show that the non-linear model is significantly better than the linear. The equations of the fitted models are:
Square root-Y logarithmic-X:
The non-linear model allows estimating the number of death cases with model fitting more than 80% (Supplementary Fig. S10).
Further, we looked at the detection limit of wastewater monitoring related to the number of positive RT-qPCR cases within a 12 days shift. Individually, the detection limit of WWTP Bratislava - Vrakuna is 1 RT-qPCR positive case per 25,000 people on 15/3/2021, while Bratislava - Petrzalka reached 1 per 4,808 on 19/1/2021. The results are also limited by reporting the positive RT-qPCR cases as separate data for each city district has been available since 10/11/2020. For Bratislava the detection limit was 1 per 8,099 on 29/9/2020 when both WWTPs were combined. It is important to stress that the results are related and strongly biased to reported RT-qPCR positive cases which vary between the countries, their testing capacities and contact tracing. In comparison, the study by Ahmed et al., could not detect SARS-CoV-2 in wastewater until 100 reported cases per 100,000 people. On the contrary, Medema et al., was capable of detecting SARS-CoV-2 in the wastewater before the first confirmed results in the monitored area 26,27. Another reason for such contrasting results is probably the different quality of the wastewater and various methodological approach influencing the detection limit. Each sewer has unique properties such as temperature, pH, presence of chemical substances and biological composition. All of these factors play a role in the degradation of viral particles. Therefore investigation of these properties is crucial and each methodological approach to detect SARS-CoV-2 from wastewater is unique for a given sewer system. However, some unification of the methodology would be vital for future comparison of the results.
Until now there was no confirmed case of infection from wastewater thus oral-fecal route is unlikely. The reason may be the inactivation of SARS-CoV-2 by gastrointestinal fluids or an unfavorable wastewater environment 15,28. The amount of the virus shed to the feces may vary in time as well as between the patients 22. Once the virus or its particles enter the sewage system, it is diluted by other types of water (industrial, rainfalls, water from snow melting, etc.) and is exposed to various physical and chemical factors. So far, viral RNA appears to be stable until reaching the primary settling tank of WWTP. Compared to non-enveloped viruses, SARS-CoV-2 has an affinity to wastewater solids, so a portion of the virus is probably sorbed on sewage walls and later to primary sludge 29. On the contrary, wastewater surveillance offers several benefits once we have a working monitoring system. In September 2020, Larsen and Wigginton published in Nature Biotechnology that theoretically, wastewater surveillance can be in 7 days lead before rapid diagnostics tests and 13-15 days before delayed diagnostic tests 1. This is in agreement with our observations where results from wastewater dated to the day of sampling were in 14 days (2 weeks) lead before standard clinical RT-qPCR testing. If we add 1-2 days of sample pretreatment and analysis we are reaching for 12-13 days advance before reported positive RT-qPCR tests. Novelty in the presented study is that a similar correlation can be found with death reported in 28 days (4 weeks) after the detected increase of viral particles in the wastewater. Although we can not specify the number of infected persons, observation of trends in near real-time can help us understand community transmission. In communities with low capacity of clinical testing and delays of diagnostics wastewater surveillance can be a temporary solution. Moreover, as it is relatively cost-effective and less invasive, wastewater monitoring can be used in low-income countries 30. However in communities with working traditional testing, data obtained from wastewater are only additional information in controlling pandemics. They can be used to check the reliability of the introduction of novel technologies or protocols.
For successful monitoring of SARS-CoV-2 in the wastewater on a larger scale, public authorities must realize the importance of possible scalability of the method 31. National agencies should show interest and provide financial and material support for research teams and water companies in the form of grants. Monitoring of COVID-19 and its mutations will require modification of the methods when compared to the monitoring of drugs or metabolites 31. Optimistic presumption for successful cooperation is the fact that across Europe there is already a working consortium of institutes and universities focusing on monitoring illegal drugs in the wastewaters 32,33. European Commission at the beginning of March created the HERA incubator. One of the focuses is the systematic surveillance of the wastewaters, including genetic sequencing at WWTPs with a connected population of over 150,000 individuals 34.
In the presented study, we detected RNA SARS-CoV-2 in wastewaters and displayed mathematical correlations between tested wastewater samples, positive RT-qPCR tests and death cases in Bratislava, Slovakia. The obtained results and subsequent mathematical modeling will be able to serve in the future as an early warning system for the occurrence of a local site of infection and at the same time will allow to predict the load on the health system up to two weeks in advance. Because each wastewater has its own characteristics (pH, temperature, (bio)chemical composition, etc.), it is necessary to approach this in other monitored localities when taking and processing samples, evaluating the results and creating the appropriate mathematical model.