key: cord-0706578-uwplrdj8 authors: Barril, Patricia Angélica; Pianciola, Luis Alfredo; Mazzeo, Melina; Ousset, María Julia; Jaureguiberry, María Virginia; Alessandrello, Mauricio; Sánchez, Gloria; Oteiza, Juan Martín title: Evaluation Of Viral Concentration Methods For Sars-Cov-2 Recovery From Wastewaters date: 2020-11-28 journal: Sci Total Environ DOI: 10.1016/j.scitotenv.2020.144105 sha: d7f81df299c043a29a612e2396d2a4735729d05d doc_id: 706578 cord_uid: uwplrdj8 Wastewater-based epidemiology (WBE) is a useful tool that has the potential to act as a complementary approach to monitor the presence of SARS-CoV-2 in the community and as an early alarm system for COVID-19 outbreak. Many studies reported low concentrations of SARS-CoV-2 in sewage and also revealed the need for methodological validation for enveloped viruses concentration in wastewater. The aim of this study was to evaluate different methodologies for the concentration of viruses in wastewaters and to select and improve an option that maximizes the recovery of SARS-CoV-2. A total of 11 concentration techniques based on different principles were evaluated: adsorption-elution protocols with negatively charged membranes followed by polyethylene glycol (PEG) precipitation (Methods 1-2), PEG precipitation (Methods 3-7), aluminum polychloride (PAC) flocculation (Method 8), ultrafiltration (Method 9), skim milk flocculation (Method 10) and adsorption-elution with negatively charged membrane followed by ultrafiltration (Method 11). To evaluate the performance of these concentration techniques, feline calicivirus (FCV) was used as a process control in order to avoid the risk associated with handling SARS-CoV-2. Two protocols, one based on PEG precipitation and the other on PAC flocculation, showed high efficiency for FCV recovery from wastewater (62.2 % and 45.0 %, respectively). These two methods were then tested for the specific recovery of SARS-CoV-2. Both techniques could recover SARS-CoV-2 from wastewater, PAC flocculation showed a lower limit of detection (4.3 × 102 GC/mL) than PEG precipitation (4.3 × 103 GC/mL). This work provides a critical overview of current methods used for virus concentration in wastewaters and the analysis of sensitivity for the specific recovery of SARS-CoV-2 in sewage. The data obtained here highlights the viability of WBE for the surveillance of COVID-19 infections in the community. al., 2020). Some researchers concentrate and purify viral particles using ultrafiltration devices (Medema et al., 2020) . Other groups had some success by filtrating the viral particles through electronegative membranes (Ahmed et al., 2020a) . Different protocol variants of polyethylene glycol (PEG) precipitation are also used for viral concentration (Ahmed et al., 2020a; Wu et al., 2020) . Another simple procedure reported is the ultracentrifugation of the wastewater, but it requires an equipment that is not often available in many laboratories (Wurtzer et al., 2020; Prado et al., 2020) . Finally, aluminum-driven flocculation has consistently detected SARS-CoV-2 RNA in sewage samples when communicated cases in those regions were only incipient or not declared at all (Randazzo et al., 2020a; Randazzo et al., 2020b) . The goal of our investigation was to evaluate different methods for the concentration of viruses in wastewater in order to select and improve a concentrating option that maximizes the recovery of SARS-CoV-2 in this complex matrix. March and the 20th of October 2020, from a wastewater treatment plant (WWTP) located at Neuquen city, in the province of Neuquen, Argentina. Two and a half liters of grab samples were collected from the WWTP influent, immediately stored at 4 °C, and dispatched to the Center of Research and Technological Assistance to the Industry associated with human illnesses (Radford et al., 2007; Mattison et al., 2009) . Briefly, subsamples of 200 mL of two sewage samples were seeded with 1.2 × 10 5 PCR units of FCV and subjected to viral concentration. One PCR Unit was defined as the last dilution of a sample from which the FCV genome could be amplified. Thus, the titer of viral RNA in a sample was the reciprocal of that dilution. One sewage sample was used for the first round of analysis, and the other sample for the duplicate. FCV was concentrated by duplicate from the seeded sewage samples using eleven previously published methods, with modifications as noted. These methods are referred as Methods 1-2 (adsorption-elution based protocols with negatively charged membranes followed by PEG precipitation), Methods 3-7 (PEG precipitation protocols), Method 8 (aluminum polychloride flocculation, PAC), Method 9 (ultrafiltration), Method 10 (skim milk flocculation) and Method 11 (adsorptionelution with negatively charged membrane followed by ultrafiltration) ( Table 2) . Method 1 (Katayama et al., 2002) began with the filtration of the sewage sample through a 0.2 μm pore-size membrane (Merck Millipore Ltd) to remove bacterial cells and debris. Then MgCl 2 was added to a final concentration of 2.5 mM and pH was adjusted to 3.5. The conditioned sample was then passed through a 0.45 μm negatively charged membrane (Merck Millipore Ltd). Subsequently, 0.5 mM H 2 SO 4 (pH 3.0) was passed through the membrane to remove cations prior to viral elution with 3 mM NaOH (pH 10.5). For neutralization, 50 μL of 100 mM H 2 SO 4 (pH 1.0) and 100 μL 100 X Method 2 is a modified version of Method 1. Briefly, the sewage sample was centrifuged at 5,000 × g for 5 min before membrane filtration. Then, as in Method 1, supernatants were passed through 2 μm membranes, conditioned with MgCl 2 and passed through a 0.45 μm negatively charged membrane. Subsequently, 0.5 mM H 2 SO 4 (pH 3.0) was passed through the membrane to remove cations prior to viral elution with Tris Glycine 1 % Beef Extract buffer (pH 9.5). The eluate was stirred for 20 min at room temperature to release the membrane-adsorbed viruses and then pH was adjusted to 7.0. Viruses were further concentrated by PEG precipitation as in Method 1. Method 3 began with the filtration of sewage through a 0.2 μm membrane. Then, the eluate was concentrated by PEG precipitation by adding PEG 6000 to a final concentration of 8 % (w/v) and NaCl to 0.3 M. Immediately after that, the sample was centrifuged at 12,000 × g for 2 h. The PEG-containing supernatant was discarded and the pellet was suspended in 300 μL lysis buffer from the Direct-zol RNA MiniPrep Kit (Zymo Research) for further RNA extraction. In Method 4 (Lewis and Metcalf, 1988) sewage was centrifuged at 4,750 × g for 20 min at 4 °C. Supernatant (S1) was maintained at 4 °C to be used later and the sediment was mixed with 3 % Beef extract / 2 M NaNO 3 eluant (pH 5.5) and stirred for 1 h at 4 °C. Solids were then removed by centrifugation at 10,000 × g for 20 min and the eluate was mixed with the first supernatant obtained (S1) and adjusted to pH 7.2. PEG 6000 was added to a final concentration of 10 % (w/v) and NaCl to 2 % (w/v). The resulting suspension was stirred for 2 h at 4 °C and centrifuged at 10,000 × g for 25 min. The PEG-containing supernatant was discarded and the pellet was suspended in 2 mL PBS (pH 7.2), adjusted to pH 8.0, incubated for 1 h with occasional vortex, and centrifuged at 10,000 × g for 20 min. The supernatant was stored at -70 °C. Prior to viral concentration steps of Method 5 (Kocamemi et al., 2020), sewage was J o u r n a l P r e -p r o o f Journal Pre-proof shaken at 4 °C at 100 rpm for 30 min to transfer viruses to the aqueous phase. Then, bacterial debris and large particles were removed from the samples by centrifugation at 7,471 × g for 30 min at 4 °C. Supernatant was filtered through a 0.45 μm membrane to remove remaining particles and the filtrate was mixed thoroughly with PEG 6000 (10 % w/v) and NaCl (0.3 M) by shaking for 1 min. The mixture was incubated at 4 °C at 100 rpm for at least 2 h. Following incubation, viruses were precipitated by centrifugation at 7,471 × g for 2 h at 4 °C. Supernatant was removed carefully and pellets were suspended with 200 μL water. The supernatant was stored at -70 °C. Method 6 (Iwai et al., 2009; Thongprachum et al., 2018) began with the addition of 16 g PEG 6000 (8 % w/v) and 4.6 g NaCl (0.4 M) to the sewage. The suspension was stirred at 4 °C for 2 h and then centrifuged at 10,000 × g for 30 min at 4 °C. The pellet was suspended in 2 mL of RNase-free distilled water and stored at -70 °C. Method 7 (Lewis and Metcalf, 1988; Greening et al., 2002) began with the adjustment of pH to 6.5 -7.2 and the addition of PEG 6000 (10% w/v) and NaCl (0.3 M). The solution was stirred for 2 h at 4 °C and then centrifuge at 10,000 × g for 25 min at 4 °C. The PEG-containing suspension was discarded and the pellet was suspended in 1 mL PBS (pH 7.2). Then, pH was adjusted to 8.0 and the solution was incubated at room temperature for 1 h with occasional agitation. After incubation, the suspension was centrifuged at 10,000 × g for 20 min and the supernatant was stored at -70 °C. In Method 8 (Randazzo et al., 2020a) an Al(OH) 3 precipitate was formed by adding run in duplicate in two independent assays. concentration method was calculated based on the copies quantified by RT-qPCR as follows: Recovery Efficiency (%) = (Virus recovered / Virus seeded) × 100 The mean and standard deviation for each concentration method was calculated. The one-way analysis of variance (ANOVA) was used to determine whether there was a difference in FCV recovery among the concentration methods analyzed. As preliminary screening of methods, eleven different protocols were initially evaluated. The mean efficiencies of the viral concentration methods tested for FCV recovery varied from 0 % to 62.2 % (Table 1) , with an average of 10.7 %. Methods 7 (a PEG precipitation approach) and 8 (based on PAC flocculation) revealed media recovery rates above 40 %, Method 4 achieved a recovery rate of 9.9 % and the other methods had media efficiency yields ≤ 1 %. As Method 7 and 8 performed statistically better than the other evaluated methods (P<0.05), SARS-CoV-2 was recovered from the seeded sewage samples after viral concentration with both methods. In order to determine the limit of detection of SARS-the concentration efficiency. The use of a non-enveloped virus such as FCV, provides an estimation of the recovery efficiency of the methods and allows to evaluate whether the protocols worked correctly, but further evaluation with SARS-CoV-2 should be carried out. Based on the equipment available in our laboratory, eleven different methods were evaluated to concentrate viruses in wastewater. Three of the tested methods were efficient to recover FCV from sewage, two based on PEG precipitation (Methods 4 and 7) and the other on PAC flocculation (Method 8). Of these three methodologies, Methods 7 and 8 showed mean recovery efficiencies higher than 40 %. Concentration methods based on viral filtration through charged membranes were not efficient for FCV recovery. It must be pointed out that high particulate and dissolved constituents present in the samples plugged the filters and more than 2 charged membranes were necessary for concentrating a 200 mL untreated sewage sample, even when pre-filtration or centrifugation steps were added in order to decrease the organic matter present in the samples. Our results disagree with Ahmed et al. (2020a) who have successfully used charged membrane filtration to recover murine hepatitis virus, a human coronavirus surrogate in sewage. The difference in the recovery yields may be related to the volume of sample processed. Larger volumes of wastewater were analyzed in this study, which resulted difficult to filter due to membrane clogging. Also, although no total inhibition of the RT-qPCR tests were noted in our study, the processing of larger volumes of samples could lead to the co-concentration of greater quantities of inhibitory substances and ultimately impact in the recovery yields. to electronegative membranes than non-enveloped viruses, like FCV (Haramoto et al., 2009; Ye et al., 2016) . However, Randazzo et al. (2020a) reported similar recovery yields for mengovirus (a non-enveloped virus) and porcine epidemic diarrhea virus (an enveloped virus member of the Coronaviridae family) when processing the samples by aluminum hydroxide adsorption-precipitation, and also their results are in line with the mengovirus recoveries reported for other concentration methodologies. In the present study, we could not recover FCV by ultrafiltration using a centrifugal concentration device. It is probable that the high concentration of particulate matter could interfere with the viral concentration, although a centrifugation and viral elution steps were added before ultrafilter centrifugation. Moreover, it was also reported by others that viral recovery efficiency varied greatly based upon the centrifugal concentration device utilized and that not all centrifugal devices can effectively concentrate SARS-CoV-2 from wastewater (Ahmed et al., 2020a). Methods 7 (a PEG precipitation approach) and 8 (based on PAC flocculation), which showed the best yields for FCV recovery from wastewater, were tested for the specific concentration of SARS-CoV-2. PAC flocculation showed a limit of detection of 4 × 10 2 GC/mL of SARS-CoV-2 in sewage, and the PEG precipitation approach revealed a limit of detection of 4 x 10 3 GC/mL. However, recoveries obtained by both concentration methods were variable, suggesting that the quantitative analysis is difficult and somewhat random. Although PAC flocculation showed a better efficiency for SARS- Table 3 . 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