FOCUS on Covid-19 Diagnosis by Wastewater-Based Epidemiology: SARS-CoV-2 Concentration and Detection Methods, Biomarkers and Future Perspectives
In April 2020, our last Editor´s Choice on Covid-19 outbreak was released. A selection of the first accepted papers including temperature and air quality influence, hospital disinfection and sewage as indicator of the impact of the virus among others were highlighted . Such selection did help to better understand this new global epidemic from an environmental and health perspective. Since then STOTEN did receive over 1000 submissions for the Covid-19 Special issue and we did publish already over 100 papers. In this summer 2020 Editor´s choice our second selection on the same topic we focus on the so- called Wastewater-Based Epidemiology (WBE) concept to monitor Covid-19 outbreak.
WBE can be used as early detection system and to determine the scale of Covid-19 outbreak . Detection of SARS-CoV-2 is generally carried out with nucleic acid–based polymerase chain reaction (PCR) assay, used for confirmation of COVID-19 patients around the globe. PCR has high sensitivity and specificity, requirements for complicated sample handling in the laboratory, skilled personnel, and a period of data processing and analysis between 4-6 hours.In wastewaters the virus causing COVID-19 (via feces introduced in waste water) can survive from hours to days. Detection of SARS-CoV-2 in sewage has been employed as a complementary method to clinical test and as early warning indicator of virus spreading in communities, covering both symptomatic and asymptomatic cases.
a. First Detection of SARS-CoV-2 in Wastewaters Around the Globe
The first group of selected seven papers report first data on COVID-19 in sewage for a variety of countries using different PCR platforms, among them Italy [1, 2], Australia [3] ,Japan [4] , USA [5], , Ecuador [6] and India[7]. The idea behind this WBE is easy to understand: patients with COVID-19 in their gastrointestinal tract will thus excrete it in their feces. It has been reported [8] that between 15 to 83% of patients infected with SARS-CoV-2 have detectable viral RNA in feces and they may continue to remain positive in the stool, even when respiratory tract samples become negative. Conversely, urine is often negative. It is worth to highlight few findings from the various studies. SP Scherchan et al., [5] reported that no one of the secondary and final treated effluents samples with chlorine were tested positive for SARS-CoV-2. In this sense P Foladori et al [8] did summarize that disinfection technologies based on hypochlorous acid can be implemented for inactivation of SARS-CoV-2 in wastewater SG Rimoldi [2] detected SARS-CoV-2 RNA in raw, but not in treated wastewaters (four and two samples, respectively, sampled in two dates). Viral RNA was not detected in the Milano and Monza WWTPs plants, equipped with tertiary treatments suggesting that additional treatment was able to remove the virus. Another study did report the levels of SARS-CoV-2 in countries with low level of sanitation, L.Guerrero-Latorre [6] detected SARS-CoV-2 in urban rivers of Quito, Ecuador, receiving direct discharges of wastewaters. Such levels were high and did match those of other cities with over 4000 hospitalized cases. Surprisingly in Quito [6] only 750 Covid-19 clinical cases were reported, suggesting a lack of PCR-based diagnosis. We can explain this discrepancy by the fact the Ecuador is a low sanitation country, with not many facilities to perform the clinical tests in one side and also to a high level of asymptomatic cases, as reported for other cases. One of the most recent examples of a first study in a large country has been recently published by M. Kumar et al., [7]. It is first WBE surveillance in India using RT-PCR and it was performed at the Old Pirana Waste Water Treatment Plant (WWTP) at Ahmedabad, Gujarat, which has 106 million liters per day and receives effluent from Civil Hospital treating COVID-19 patients. Several genes like ORF1ab, N and S genes of SARS-CoV-2 were detected only in the influent with no genes detected in effluent . Increasing levels of SARS-CoV-2 genetic loading in the wastewater did correspond to an increase in the number of active COVID-19 patients in the city. The number of gene copies was comparable to those reported in untreated wastewaters of Australia reported by W.Ahmed et al.,[3] .
b. Persistence and Virus Concentration and Detection Methods
Survival and/or persistence of the virus in the wastewater environment is a key parameter when considering SARS-CoV-2 detection in WBE. M. Arslan et al., [9] looked at the possibilities of transmission routes of SARS-CoV-2 and SARS-Cov-1 via fecal-oral and aerosol-borne and its persistence in wastewaters . based on present knowledge coronaviruses have, in general, low stability in the environment due to the natural action of oxidants that disrupt the viral envelope. Persistence in waste water is driven by several parameters such the presence of organic matter oxidants and temperature. For instance at 20 oC SARS-CCoV-1 was found to survive for 2 days in domestic sewage but at lower temperatures of 4 oC it can persists up to 14 days. They also reported that previous recommended dosage of chlorination has been found to be not sufficient to inactivate SARS-CoV-2 in places where viral load is high such as hospitals.
There is a comprehensive list concentration methods used on the RNA extraction protocol in WBE that need to be used before PCR detection like electropositive or electronegative membranes , ultrafiltration, polyethylene glycol (PEG) precipitation and separation , ultracentrifugation, skimmed-milk flocculation, ultrafitration, monolithic adsorption filtration columns among others. Generally similar protocols for the influent, secondary-treated sewage are used and a volume of 100mL of untreated wastewater samples is sufficient to detect enteric viruses . Most of the concentration methods were developed for non enveloped enteric viruses such as norovirus, and hepatitis A virus among others. Excellent review papers on concentration methods of viruses in WBE were recently published by W.Ahmed et al., [10] and D.Lu et al., [11] .
D. Lu [11] did highlight that PEG-based separation method is the most used for the COVID-19 in WBE. The authors indicated as well that the electronegative membrane filtration method may have problems with the preferential adsorption of organic matter on the charged membrane surface and the potential risk of clogging when handling turbid wastewater samples.
Analysis of SARS-CoV-2 in wastewaters by different PCR platforms is as well matter of concern. To investigate a bit more in this direction G. La Rosa and co-workers [ 1 ] did analyze the presence of SARS-CoV-2 using three different nested RT-PCR assays and one real-time qPCR assay. Primers were also indicated to be very relevant using this methodology. From all the different methods used. a novel nested PCR assay specific for SARS-CoV-2 detection was proposed in this case. MVA Corpuz et al [12] presents an updated and comprehensive review on the different methods used for detection and quantification of viruses in WWTPs, including both wastewater and sludge. Recent efforts to improve efficiency of virus detection and quantification methods in the complex wastewater and sludge matrices are highlighted .
Novel approaches to detect SARS-CoV-2 in WBE digital PCR (dPCR) need attention as recently reported by W. Ahmed et al. [13]. The major benefit of dPCR over qPCR is the direct absolute quantification of virus genome copy numbers in a sample without the necessity of external calibration. dPCR platforms can generally be divided into two groups: droplet dPCR (emulsion based) and chip-based dPCR (microfluidic). Digital PCR (dPCR) is at least 10-fold lower than that of RT-qPCR The major advantage of dPCR over qPCR is that it performs absolute quantification, and hence, no standards are required.
c.Biomarkers
To this end, the solution at present to monitor SARS-CoV-2 in WBE could be a combination of technologies and methodological strategies already in place such as PCR and endogenous biomarkers measurements using ELISA and or MS. Why we do not use both approaches for WBE?. Certainly both methods got advantages and disadvantages. Papers selected in this Editor´s choice did discuss a lot already about PCR standardization. We are also aware that inflammatory response biomarkers are related to SARS-CoV-2 but also to other diseases. Both methods suffer from drawbacks but obviously they are complementary too. On top of that WBE seems to detect more possible cases of patients than clinical tests, including asymptomatic ones, and also other ones who did recover from Covid-19. In this sense WBE can provide additional information not only on asymptomatic cases but also on immunized patients who did recover from Covid-19 . Based on these data, epidemiologists could be able to estimate if Covid-19 outbreak would become like a common flu in the near future.
Biomarkers discharged either through urine or feces, are of interest because they can be rather specific for given infectious diseases and good candidates for WBE. A likely place to start evaluating the use of biomarkers would be to make use of the fact that Covid-19 can involve extensive inflammatory damage. The archetype biomarker for systemic oxidative stress is the prostaglandin-like class of substances called isoprostanes as reported by CG Daughton [14] .This paper summarizes that testing for biomarkers might have several other major advantages over the use of PCR such as biomarkers might be more universally excreted among infected individuals, the excreted levels can track the severity of infection, biomarker testing might have tighter ranges for per-capita excretion, facilitating better calibration and more accurate estimations of the number of infected individuals in a community, and avoiding a potential under-appreciated problem with using PCR.
Another advantage of using biomarkers instead of SARS-CoV-2 with PCR is on the detection side since most of the measurements for biomarkers molecules are carried out by mass spectrometry(MS) or ELISA . It is well-known that MS or ELISA provide better accuracy and detection limits and validation of results as compared to standard PCR measurements.
With this said, the following question will needs answer : Can we look for the same type of biomarkers,. i.e inflammatory response biomarkers already used in clinical diagnostics for the detection of Covid-19 outbreak in WBE?
d.Final remarks and Future Perspectives
PCR is still the most widely used method for SARS-CoV-2 detection in waste waters as well as in clinical studies. One of the problems is the complexity of the wastewater matrix that needs to be treated by using different concentration methods . Efforts are certainly needed to evaluate the applicability of these existing methods to concentrate SARS-CoV-2. As recommended by W.Ahmed et al [13] there is an urgent need to evaluate RT-qPCR methods used by different laboratories with a clear target to achieve a verification/standardization agreement. Several factors such as qPCR platforms, PCR inhibitors, nucleic acid extraction efficiency and low levels of targets may have contributed to the observed discrepancies between laboratories .Standardization of the whole protocol is needed to recover and detect SARS-CoV-2 from environmental water samples, including all steps : concentration method, PCR assay, and process controls.
With this said, the expected future of PCR in WBE will greatly benefit by incorporating new technological developments from the clinical field such as digital PCR reported by W. Ahmed et al., [13]. References in the literature highlight the need of paper based devices and smartphones already used in clinical testing look like a reasonable alternative. Can smartphome technology be used for detecting viruses in wastewaters? MVA Corpuz et al ., [12] reported an example from the literature using a smart-phone fluorescence microscope and a mobile graphical user interface. Such methodology was applied to the detect specific of noroviruses with antibody-based method.
That being said, I hope that this Editor´s choice selection will help to better understand and monitor the Covid-19 outbreak using WBE. And most importantly, in these strange times, stay safe and enjoy this uncommon and highly needed 2020 summer break!
References
G La Rosa, M Iaconelli, P. Mancini, G. Bonanno Ferraro, C. Veneri, L. Bonadonna, L. Lucentini, E. Suffredini, First detection of SARS-CoV-2 in untreated wastewaters in Italy, Science of the Total Environment, 736 (2020)139652. S. G. Rimoldi, F. Stefani, A. Gigantiello,S. Polesello, F. Comandatore, D. Mileto,M. Maresca, C. Longobardi, A. Mancon,F. Romeri, C. Pagani, F, Cappelli, C. Roscioli, L. Moja, M.R. Gismondo, F. Salerno, Presence and infectivity of SARS-CoV-2 virus in wastewaters and rivers, Science of the Total Environment, 744 (2020) 140911.W. Ahmed, N. Angel, J. Edson, K. Bibby, A. Bivins, J. W. O'Brien, Ph. M. Choi, M. Kitajima, S.L. Simpson, J. Li, Ben Tscharke, R. Verhagen, W.J.M. Smith, J, Zaugg, L. Dierens, Ph. Hugenholtz,K. V. Thomas, J, F. Mueller, 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, Science of the Total Environment, 728, 2020, 138764.E. Haramoto , B. Malla , O. Thakali , M. Kitajima , First environmental surveillance for the presence of SARS-CoV-2 RNA in wastewater and river water in Japan, Science of The Total Environment, 737 (2020)140405.S.P Sherchan, S. Shahin, L. M. Ward, S. Tandukar, Tiong G. Aw, B. Schmitz, W. Ahmed and M. Kitajima, First detection of SARS-CoV-2 RNA in wastewater in North America: A study in Louisiana, USA, Science of the Total Environment, 743 (2020) 140621.L. Guerrero-Latorre, I. Ballesteros, I. M. Villacrés, M.Genoveva Granda, B. Freire-Paspuel, B. Ríos-Touma, SARS-CoV-2 in river water: Implications in low sanitation countries, Science of the Total Environment, 743 (2020)140832.M. Kumar, A. K.Patel, A. V Shah, J. Raval, N. Rajpara, M. Joshi ,C. G Joshi First proof of the capability of wastewater surveillance 1 for COVID-19 in India through detection of genetic material of SARS-CoV-2, Science of the Total Environment,P. Foladori, F. Cutrupi, N. Segata, S. Manara,F. Pinto, F. Malpei, L. Bruni and G. LaRosa, SARS-CoV-2 from feces to wastewater treatment: What do we know? A review, Science of the Total Environment , 743 (2020) 140444.M Arslan, B. Xub, M.Gamal El-Din, Transmission of SARS-CoV-2 via fecal-oral and aerosols–borne routes:Environmental dynamics and implications for wastewater management in underprivileged societies, Science of the Total Environment, 743 (2020) 140709.W. Ahmed , P. M. Bertsch , A. Bivins , K. Bibby , K. Farkas , A. Gathercole ,Eiji Haramoto , Pradip Gyawali , Asja Korajkic , Brian R. McMinn, Jochen F. Mueller , Stuart L. Simpson ,Wendy J.M. Smith , Erin M. Symonds , Kevin V. Thomas , Rory Verhagen , Masaaki Kitajima , Comparison of virus concentration methods for the RT-qPCR-based recovery of murine hepatitis virus, a surrogate for SARS-CoV-2 from untreated wastewater, Science of the Total Environment, 739 (2020)139960.D. Lu, Z. Huang, J. Luo, X. Zhang, S. Sha, Primary concentration – The critical step in implementing the wastewater based epidemiology for the COVID-19 pandemic: A mini-review, Science of the Total Environment,https://doi.org/10.1016/j.scitotenv.2020.141245M.V A. Corpuz, A. Buonerba, G. Vigliotta, T. Zarra, F. Ballesteros, P. Campiglia,V. Belgiorno, G. Korshin, V. Naddeo, Viruses in wastewater: occurrence, abundance and detection methods Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2020.140910W. Ahmed , S. Payyappat , M. Cassidy , N. Harrison, C. Besley Interlaboratory accuracy and precision among results of three sewage-associated marker genes in urban environmental estuarine waters and freshwater streams, Science of the Total Environment, 741 (2020)140071.CG. Daughton, Wastewater surveillance for population-wide Covid-19: The present and future, Science of the Total Environment, 736 (2020)139631.
