key: cord-0962038-1caehu58
authors: Varbanov, M.; Bertrand, I.; Philippot, S.; Retourney, C.; Gardette, M.; Hartard, C.; Jeulin, H.; Duval, R. E.; Loret, J.-F.; Schvoerer, E.; Gantzer, C.; Bertrand, Isabelle; Gantzer, Christophe; Le Guyader, Soizick; Maday, Yvon; Maréchal, Vincent; Mouchel, Jean-Marie; Moulin, Laurent; Boni, Mickael; Wurtzer, Sébastien
title: Somatic coliphages are conservative indicators of SARS-CoV-2 inactivation during heat and alkaline pH treatments
date: 2021-07-15
journal: Sci Total Environ
DOI: 10.1016/j.scitotenv.2021.149112
sha: 45b69977fb1b1159d3829432551ec83773c7201c
doc_id: 962038
cord_uid: 1caehu58

High concentrations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome have been described in wastewater and sewage sludge. It raises the question of the security of land sludge disposal practices during a pandemic. This study aimed to compare SARS-CoV-2's resistance to the main inactivating factors in sludge treatments, pH and heat, to that of native wastewater somatic coliphages. The latest can be easily used as an indicator of treatment efficiency in the field. The effects of heat treatment and pH on the survival of SARS-CoV-2 and somatic coliphages were investigated in simple media. The T90 value (time required for a 90% reduction in the virus or a 1 × log10 decline) at 50 °C was about 4 min for infectious SARS-CoV-2, and around 133 min for infectious somatic coliphages, with no decrease in SARS-CoV-2 genome. For infectious SARS-CoV-2, a slight decrease (<1 log10 unit) was observed at pH 9 or 10 for 10 min; the decrease was over 5 log10 units at pH 11. However, both SARS-CoV-2 genome and infectious somatic coliphages decreased by less than 1 log10 unit at pH 12. All thermal or pH-based treatments that can remove or significantly reduce infectious somatic coliphages (>4 log10) can be considered efficient treatments for infectious SARS-CoV-2. We concluded that somatic coliphages can be considered highly conservative and easy to use indicators of the inactivation of SARS-CoV-2 during treatments based on heat and alkaline pH.

Wastewater treatment plants produce large quantities of sewage sludge. The sludge may be incinerated or reused in agricultural applications as a fertiliser. However, raw sludge contains a high quantity of highly diverse pathogenic microorganisms including parasites, bacteria, and viruses (Viau et al., 2011) . The main antiviral treatments usually target enteric viruses because of their high degree of resistance to this environment. Enteric viruses are small viruses (20-90 nm in diameter) whose genome is protected only by a proteic capsid.

They replicate in the human gut, causing various pathologies (e.g. gastroenteritis, hepatitis, meningitis, heart damage, etc.).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for the coronavirus pandemic that has been raging since December 2019, is an enveloped virus, with a helical nucleocapsid surrounded by lipids. This virus is 60-140 nm in size and its genome is composed of a single RNA-positive strand of around 30 kb (Foladori et al., 2020) .

SARS-CoV-2 provokes acute pneumonia (cough, fever, rhinorrhoea, dyspnoea) and multiple extra-respiratory signs and, importantly, has also been demonstrated to cause gastrointestinal symptoms (Gu et al., 2020; Pan et al., 2020; Yeo et al., 2020; Zhou et al., 2020) . SARS-CoV-2 can productively replicate in human gut enterocytes (Lamers et al., 2020) and the SARS-CoV-2 genome has frequently been detected in the stools of both symptomatic and asymptomatic patients Wölfel et al., 2020; Xu et al., 2020; Zhang et al., 2020) . It is therefore unsurprising to find high concentrations (until 3 x the risk due to the land disposal of sludge into 3 transmission factors: direct contact, aerosol, and ecological transmission . Given that aerosols are one of the main modes of transmission (Viau et al., 2011) , caution should clearly be taken with SARS-CoV-2, which is a respiratory virus.

Sludge is usually treated using several processes that allow for the complete removal of infectious forms of pathogenic microorganisms (class A biosolids) or their partial removal (class B biosolids) (Viau et al., 2011) . The main treatments that inactivate the virus are primarily based on heat and pH . Temperatures applied during sludge treatments can vary widely depending on treatment type: 35-40°C in mesophilic digestion, almost 50-55°C in thermophilic digestion, over 55-65°C in composting, and up to 70°C in pasteurization (Alkarimiah and Suja, 2019; Viau et al., 2011) . Lime stabilisation leads to a huge increase in pH: it usually raises the pH to higher than 11 and ideally to 12 (Parmar et al., 2001) . Viau et al. (2011) have emphasised that a rigorous biosolid pathogen process is the key to reducing pathogen exposure and the risk of infection. Foladori et al. (2020) have reported on the lack of studies on how SARS-CoV-2 behaves in sludge.

The direct detection of SARS-CoV-2 genomes in sludge is not an appropriate way of evaluating treatment efficiency. The presence of the genome does not prove the presence of the infectious virus, since genomes can persist after the virus is no longer infectious. This has been demonstrated to be true of a wide spectrum of viruses and treatments (Gassilloud et al., 2003; Hartard et al., 2018) . The presence of viral genome in biosolids may not be a reliable indicator of the presence of the infectious pathogen (Viau et al., 2011) . There have been reports of very high concentrations of SARS-CoV-2 genome in stool and wastewater samples, although very few studies have demonstrated the presence of infectious particles (Foladori et al., 2020) . However, the absence of the SARS-CoV-2 genome does not prove the J o u r n a l P r e -p r o o f Journal Pre-proof absence of contamination because there is no standardised method for quantifying such viruses in sludge and the very small volumes used for detection purposes result in a high detection threshold.

The gold standard for the evaluation of the efficacy of a virucidal treatment is cell culture.

SARS-CoV-2 is easily cultured on different cell lines (Park et al., 2020 ), but culture conditions in level 3 biosafety labs (BSL3) and the complexity of the environmental matrix make this an inadequate approach for evaluating sludge treatments. There is an urgent need for SARS-CoV-2 inactivation data, to allow us to evaluate the transmission risk posed by wastewater treatment plants .

Bacteriophages have been used as viral indicators of the efficiency of water and sludge treatments for years (Martin-Diaz et al., 2020) . Quantifying infectious phages is rapid (24 h) and easy. Coliphages (e.g. somatic coliphages or F-specific RNA phages) are most frequently used as their behaviour is representative of pathogenic enteric viruses. Somatic coliphages represent a structurally diverse group that is highly resistant to treatments and to heat and pH in particular. F-specific RNA phages are a homogeneous structural group similar to enteric viruses but slightly less resistant to heat and pH, as well as more resistant to other factors, such as UV light (Simonet and Gantzer, 2006) . Coliphages are naturally present in wastewater worldwide (Lucena et al., 2004) at concentrations higher than those observed for SARS-CoV-2 at the height of the pandemic (Bertrand et al., 2021) . Coliphages are found in primary sludge at concentrations of 10 5 -10 7.5 plaque-forming units (PFU)/g of dry matter (Martin-Diaz et al., 2020) . Coliphages may therefore be used as a field indicator to evaluate the virological risk associated with the reuse of sludge in agriculture in the context of the SARS-CoV-2 pandemic. However, there is a lack of comparative data on their resistance to sludge treatments.

This study was designed to assess the relative resistance of infectious SARS-CoV-2 and native somatic coliphages in wastewater to the main inactivating factors observed in sludge treatments: pH and heat. Native somatic coliphages were used to reflect their wide diversity.

They were compared using the same medium for both viruses.

Wastewater samples were collected at a wastewater treatment plant (WWTP) located in the French "Grand Est" region. The average volume of influent treated in this WWTP was ~88,000 m 3 /day (~250,000 inhabitants). Average daily samples were taken by an automated sampler following either an initial screening step to separate large solids from liquid (i.e. screened wastewater) or a sedimentation step (settled wastewater). Sampling was based on flow rate and started at 7 am on day 1, finishing at 7 am on day 2. Samples were stored in a refrigerated polyethylene tank at 5°C. The final volume collected was between 15 and 20 L depending on the flow rate of the entrance of the treatment plant. Samples were then homogenised and allowed to settle into 1 L glass bottles. Upon arrival at the laboratory, the wastewater samples were centrifuged at 6,000 × g for 20 min and the supernatant was filtered through 0.45 µm and 0.22 µm filters (Minisart® Sartorius France S.A.S., Aubagne, France) to remove microorganisms that could interfere with cell culture infectivity assays of coronaviruses.

The two different SARS-CoV-2 isolates used in this study were obtained from diagnosed patients, whose samples were investigated at the local university hospital (CHRU Brabois, Nancy, France) in compliance with ethical standards and requirements, i.e. informed consent. The nasopharyngeal samples collected in a universal transport medium (UTM) containing 1 mL of viral transport media (Copan Diagnostics Inc., Murrieta, CA, USA) were tested using reverse transcription -quantitative polymerase chain reaction (RT-qPCR) by the laboratory at the local university hospital and found to be positive for SARS-CoV-2 RNA. The 

The infectivity of the viral samples was titrated in triplicate on 96-well microtiter plates containing 100 μL of confluent Vero E6 cells. One hundred μL of serial 10-fold dilutions of the virus from 10 −1 to 10 -8 in DMEM medium with 2% FCS (i.e. maintenance medium) was added to Vero E6 cells. The infected cells were incubated at 37°C in 5% CO 2 for 7 days. The appearance of CPE was recorded daily. The tissue culture infectious dose (50%) (TCID 50 ), defined as the dilution of the virus required to infect 50% of the cell culture, was determined using the Reed and Muench method (Reed and Muench, 1938) and expressed as TCID 50 / mL.

Titrations were performed in triplicate.

Nucleic acid extraction was performed using NucliSENS® reagents (bioMérieux). Two volumes of NucliSENS® lysis buffer were added to each volume of liquid sample. Following 10 min of incubation at room temperature, nucleic acid extraction was performed using 70 µL of magnetic silica beads and the NucliSENS® easyMAG™ platform (bioMérieux). The extracted nucleic acids were eluted in 100 µL of elution buffer and stored at -80°C until needed for SARS-CoV-2 genome quantification.

SARS-CoV-2 RNA quantification was performed using E set real-time RT-PCR, developed by Corman et al. (2020) for the envelope protein (E) gene. Quantification was performed using an RNA UltraSense™ One-Step Quantitative RT-PCR system (Applied Biosystem™, Thermo Fischer Scientific) as previously described (Bertrand et al., 2021) . The real-time RT-PCR assays were performed using 5 µL of nucleic acid sample in a 25-µL reaction volume to obtain final concentrations of 0.4 µM for each primer and 0.2 µM for the probe. A CFX96 J o u r n a l P r e -p r o o f Journal Pre-proof Touch real-time PCR detection system (BioRad, Marnes-la-Coquette, France) was used for the real-time RT-PCR assays. The RT step was performed for 30 min at 50°C and PCR amplification was performed for 2 min at 95°C, followed by 45 cycles of 15 s at 95°C and 30 s at 58°C. Negative and positive controls were included in each experiment. The nCoV-ALL-Control plasmid (Eurofins genomic) quantified using Qubit 4 fluorometer (Invitrogen) was used for the standard curve which ranged from 1 × 10 -1 to 1 × 10 4 genome copies (gc)/RT-qPCR reaction. The limit of detection was between 1 and 10 gc/RT-qPCR reaction. The Cq values obtained at T 0 and time x, and the slope of the standard curve allowed us to determine the loss in genome occurring during time x.

Somatic coliphages naturally present in the sewage samples were used in these experiments.

They were quantified using the E. coli (WG5) bacterial host strain according to the standard ISO 10705-2 method (International Organization for Standardization, 2000). Concentrations were expressed in plaque-forming units (PFU)/mL.

One milliliter of virus stock (10 5 -10 6 TCID 50 /mL) was spiked into 9 mL aliquots of freshly settled wastewater. A positive control sample for measuring the initial virus concentration in DMEM with 2 % FCS, as well as negative control including settled wastewater in DMEM with 2 % FCS were included.

The samples were held at room temperature (20° C) for 7 days, without agitation for the duration of the experiment. At each time point (day 1 to 7), samples were taken and assayed for virus infectivity on Vero E6 cell line and expressed in TCID 50 / mL. In parallel, infectious somatic coliphages were quantified in wastewater every 24 h during storage at room temperature for 7 days.

Different dilutions of NaOH (1 M) solution in distilled water were prepared in order to achieve final solutions of pH 9, 10, 11 and 12. Fifty µL of stock SARS-CoV-2 virus (10 5 -10 6 TCID 50 /mL) were added to 450 µL of different dilutions of NaOH and to distilled water as a control. After 10 min of incubation at room temperature, the dilutions were neutralised to pH 7.5 using HCl (0.1 M). Separately, with the somatic coliphages, settled wastewater was diluted 10-fold in sterile, deionized water. The initial pH was 8.0. A NaOH solution (1 M) was added to produce pH values of 9-12, as with SARS-CoV-2. Following 10 min of incubation at room temperature, the solution was neutralised by adding HCl solution (1 M) until it reached pH 8. Measuring the concentrations of infectious SARS-CoV-2, SARS-CoV-2 genome and infectious somatic coliphages before (C 0 ) and after (C x ) pH treatment of the solutions stored at room temperature allowed us to determine the decay rate of each parameter.

The samples were analysed using Excel 2007 (Microsoft Corp., San Diego, CA). Regression analysis was used to model the survival of SARS-CoV-2 and somatic coliphages as a function of time. To this end, simple linear regression models were fitted to experimental data and used to estimate T 90 reduction times (predicted number of days taken to achieve a 1-log 10 reduction). The survival function of each virus under the given experimental conditions (i.e., temperature or pH) was modelled as follows: 

Heat treatment was applied to SARS-CoV-2 and somatic coliphages in triplicate.

Temperatures of 40, 50, 60 and 70°C were applied for 30 min to reflect the range of temperatures usually obtained during the treatment of sludge (Figure 1 ). The infectivity of SARS-CoV-2, somatic coliphages and SARS-CoV-2 genome before and after heat treatment was estimated. A 30 min exposure to 40°C produced almost no effect on SARS-CoV-2 or somatic coliphages that could be detected using cell culture or RT-qPCR methods. At 50°C, inactivation of 5.5 log 10 units was observed with infectious SARS-CoV-2, whereas SARS-CoV-2 genome and infectious somatic coliphages remained stable. Due to the initial concentrations of infectious SARS-CoV-2 in our 3 experiments, assessment of inactivation was limited to a maximum of 5.5 log 10 units and this level was also reached at 60 and 70°C. At 70°C, some J o u r n a l P r e -p r o o f Journal Pre-proof inactivation of somatic coliphages was also observed, ranging between 0.9 and 1.6 log 10 units (n=3). A decrease in SARS-CoV-2 genome was only observed at 70°C: its mean value was 1.5 log 10 units.

Other experiments were performed by exposing SARS-CoV-2 and somatic coliphages to 50°C for 10-60 min (Figure 2 ). The decrease in infectivity observed in SARS-CoV-2 and somatic coliphages allowed us to determine their respective T 90 values (time required for a 90% reduction in the virus or a decline of 1 x log 10 ) at this temperature. At 50°C, infectious SARS-CoV-2 had a T 90 value of about 4 min, compared with 133 min for infectious somatic coliphages. No decrease in SARS-CoV-2 genome was observed at this temperature.

The infectivity of SARS-CoV-2 and somatic coliphages was also monitored in wastewater maintained at 20°C for 7 days (Figure 3) . A constant decrease in infectivity was observed for SARS-CoV-2 and it reached at day 5 the maximum of 5.0 log 10 allowed by the initial concentration. From the decrease observed the first four days, it was possible to determine that the T90 value was about 18 h. In accordance with the results previously obtained with higher temperatures, the infectivity of somatic coliphages remained stable during the 7 days of the experiments.

A pH range of 9-12 was applied to SARS-CoV-2 and somatic coliphages for 10 min (Figure 4) .

A slight decrease (< 1 log 10 unit) in infectious SARS-CoV-2 was observed at pH 9 or 10. There was a decrease of approximately 5.5 log 10 units at pH 11. The decrease was much smaller across all other parameters. There were decreases in SARS-CoV-2 genome and somatic coliphages of less than 1 log 10 unit at pH 12.

Increasing the exposure time at pH 9 or 10 from 10 to 60 min did not affect the inactivation of infectious SARS-CoV-2 (data not shown).

We compared the inactivation of SARS-CoV-2 and somatic coliphages following thermal and alkaline treatments. The objective was to ascertain whether these bacteriophages are a useful tool for evaluating the effectiveness of SARS-CoV-2 sludge treatments.

The probability that infectious SARS-CoV-2 is present in raw wastewater is still hotly debated. Many studies have demonstrated the presence of SARS-CoV-2 genome in stools and wastewater. A review by Foladori et al. (2020) estimated that SARS-CoV-2 is present in 3.8-80% of stool samples. Viral concentration in the stools of patients, with and without diarrhoea, ranged from 5 x 10 3 -10 7.6 genome copies (gc)/mL (Cheung et al., 2020; Foladori et al., 2020) . The maximum concentration in raw wastewater was almost 10 7 genome copies/L (Wurtzer et al., 2020) . Despite these very high genome concentrations, there is very little evidence that infectious SARS-CoV-2 is present in stools or wastewater. Some studies describe the presence of infectious SARS-CoV-2 in stool without providing any details about the procedure used to obtain these results Xiao et al., 2020) . Zhang et al. (2020) used electron microscopy to obtain an image of the virus after inoculating Vero cells with stool samples, but failed to provide any quantitative data. Environmental studies by of infectious SARS-CoV-2. The worst case estimates of 10 7 gc/L SARS-CoV-2 in wastewater at the peak of the first epidemic wave in France therefore suggests a much lower concentration of infectious SARS-CoV-2.

Infectious somatic coliphages are relatively stable in raw wastewater over time. Their concentration in raw wastewater has been shown to vary between 10 7 and 10 8 PFU/L worldwide (Lucena et al., 2004) . Because of the rich diversity of these phages, the concentration of corresponding genome copies cannot be estimated. Nevertheless, the concentrations are surely much higher than the concentration of infectious SARS-CoV-2, even in the worst-case scenario.

Together, these data suggest that the concentration of infectious SARS-CoV-2 in raw wastewater may be considerably lower than the concentration of SARS-CoV-2 genome or of infectious somatic coliphages. However, although the survival rate of infectious virus may be low, we cannot completely rule out the presence of infectious SARS-CoV-2 in raw wastewater given the current state of our knowledge. We may therefore assume that infectious SARS-CoV-2 can be present in primary sludge.

The prevalence of SARS-CoV-2 in sludge has been less well described. Graham et al. (2021) found a higher concentration in sludge than in wastewater, but the concentration in sludge was still less than 10 4 gc/g. Again, we can assume that genome analysis alone will lead to a huge overestimation of the presence of infectious virus. Infectious somatic coliphages have been estimated to be present in primary sludge at concentrations of 10 5.2 -10 7.5 PFU/g of dry weight (Martin-Diaz et al., 2020) . Once again, these concentration values greatly exceed those of SARS-CoV-2, even when our estimates are based on the presence of viral genome.

Sludge treatments usually rely primarily on heat and alkaline pH to reduce microbiological contamination. The data obtained by our study provide a very clear picture of the impact of (Quist-Rybachuk et al., 2015; Leclercq et al., 2014; Darnell et al., 2004; Rabenau et al., 2005; Casanova et al., 2010; Casanova et al., 2009; Laude, 1981; Weiss and Horzinek, 1986) . This is also true of other enveloped viruses, such as Phi 6 phage and filovirus (Casanova and Weaver, 2015; Bibby et al., 2015; Piercy et al., 2010) . Mesophilic and thermophilic anaerobic sludge treatments produce inactivations of around 2 and 4 log 10 respectively after 21 days, but lead to an over 6 log 10 inactivation of Phi 6 phage in both cases (Pepper et al., 2010; Sassi et al., 2018) .

The same significant difference between SARS-CoV-2 and somatic coliphages was observed in pH treatments. At a pH of below 11, little inactivation was observed within 10 min of exposure at room temperature, as previously demonstrated with other coronaviruses, such as PEDV (porcine epidemic diarrhoea virus) (Hofmann and Wyler, 1989; Quist-Rybachuk et al., 2015) . Nevertheless, at a pH of 11 and above, decreases of 1-log 10 and over 5-log 10 were observed in somatic coliphages and SARS-CoV-2, respectively.

Synergistic effects between pH and temperature can be observed (Hofmann and Wyler, 1989) . For example, at pH 9, PEDV had a T 90 value of about 27 min at 40°C, 18 min at 44°C J o u r n a l P r e -p r o o f Journal Pre-proof and 4 min at 48°C (Quist-Rybachuk et al., 2015) . At pH 10.2, the T 90 values were around 14, 8 and 1 min, respectively (Quist-Rybachuk et al., 2015) . Some irreversible conformational changes to the viral envelope proteins that prevent cell receptor recognition may occur at an alkaline pH, as described with MHV (Weismiller et al., 1990) .

Together, our results allow us to draw important conclusions regarding sludge storage and treatments in the context of land disposal. Taking into account the rapid loss of infectivity of SARS-CoV-2 in wastewater within a few days at 20°C, and the necessarily long periods of storage of sludge before land spreading, ranging from a few weeks to several months, the presence of infectious SARS-CoV-2 in stored sludge seems very unlikely. Additionally, our results demonstrate that the methods usually applied for sludge hygienization, consisting of heat or pH treatment, will be very efficient in eliminating infectious SARS-CoV-2 particles, if any is still present in sludge. Somatic coliphages are clearly highly conservative indicators of SARS-CoV-2 inactivation. All thermal and pH-based treatments that can remove or significantly reduce somatic coliphages (e.g. ≥ 6 log 10 for a class A reclaimed water (Alcade-Sanz and Gawlik, 2017)) should be viewed as effective SARS-CoV-2 treatments.

Consequently, somatic coliphages constitute a simple, easy-to-implement and cheap surrogate to validate the effectiveness of SARS-CoV-2 treatments. 

JRC Science for Policy Report. EUR 28962 EN. Publications Office of the European Union

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

Effects of technical factors towards achieving the thermophilic temperature stage in composting process and the benefits of closed rector system compared to conventional method -a mini review

The impact of temperature on the inactivation of enteric viruses in food and water: a review

Epidemiological surveillance of SARS-CoV-2 by genome quantification in wastewater applied to a city in the northeast of France: comparison of ultrafiltration-and protein precipitation-based methods

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

Inactivation of the coronavirus that induces severe acute respiratory syndrome, SARS-CoV

SARS-CoV-2 from faeces to wastewater treatment: What do we know? A review

Presence of viral genomes in mineral water: a sufficient condition to assume infectious risk?

SARS-CoV-2 RNA in wastewater settled solids is associated with COVID-19 Cases in a large urban sewershed

COVID-19: Gastrointestinal manifestations and potential fecaloral transmission

Quantitation, biological and physicochemical properties of cell culture-adapted porcine epidemic diarrhea coronavirus (PEDV)

ISO 10705-2. Water quality: Detection and enumeration of bacteriophages

SARS-CoV-2 productively infects human gut enterocytes

Thermal inactivation studies of a coronavirus, transmissible gastroenteritis virus

Heat inactivation of the Middle East respiratory syndrome coronavirus. Influenza Other Respir

Reduction of bacterial indicators and bacteriophages infecting faecal bacteria in primary and secondary wastewater treatments

Review: Indicator bacteriophages in early stage of the epidemic in The Netherlands

Characterization of the combined effects of enzyme, pH and temperature treatments for removal of pathogens from sewage sludge

Lung recruitability in COVID-19-associated acute respiratory distress syndrome: A singlecenter observational study

Virus isolation from the first patient with SARS-CoV-2 in Korea

Pathogens and indicators in United States Class B biosolids: national and historic distributions

The survival of filoviruses in liquids, on solid substrates and in a dynamic aerosol

Sensitivity of porcine epidemic

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

A simple method of estimating fifty per cent endpoints

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

Comparative survival of viruses during thermophilic and mesophilic anaerobic digestion

Inactivation of poliovirus 1 and F-specific RNA phages and degradation of their genomes by UV irradiation at 254 nanometers

Toward a Consensus View on the Infectious Risks Associated with Land Application of Sewage Sludge

Detection of SARS-CoV-2 in Different Types of Clinical Specimens

subunits and detect a conformational change in the subunit released under mild alkaline conditions

Resistance of Berne virus to physical and chemical treatment

Virological assessment of hospitalized patients with COVID-2019

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

Evaluation of lockdown effect on SARS-CoV-2 dynamics through viral genome quantification in wastewater

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

The potential exposure and transmission risk of SARS-CoV-2 through sludge treatment and disposal

Enteric involvement of coronaviruses: is faecal-oral transmission of SARS-CoV-2 possible?

Fecal specimen diagnosis 2019 novel coronavirus-infected pneumonia

Effect of Gastrointestinal Symptoms in Patients With COVID-19

Methodology, Formal analysis, Investigation, Writing -Original Draft, Supervision, I. Bertrand: Methodology, Formal analysis, Investigation, Writing -Original Draft, Supervision, S. Philippot: Investigation, Writing -Review & Editing, C. Retourney: Investigation, Writing -Review & Editing, M. Gardette: Investigation, Writing -Review & Editing, C. Hartard: Formal analysis, Investigation, Writing -Review & Editing, H. Jeulin: Methodology, Resources, Writing -Review & Editing, Supervision, R.E. Duval: Writing -Review & Editing, Obépine: Writing -Review & Editing, Funding acquisition, J.-F. Loret: Conceptualization, Writing -Review & Editing, Funding acquisition, E. Schvoerer: Methodology, Resources, Writing -Review & Editing, Supervision, C. Gantzer: Conceptualization, Methodology

☒ 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.J o u r n a l P r e -p r o o f