key: cord-0697049-zgy1a6m5
authors: Kabdaşlı, Işık; Tünay, Olcay
title: Concentration techniques tailored for the detection of SARS-CoV-2 genetic material in domestic wastewater and treatment plant sludge: A review
date: 2021-08-30
journal: J Environ Chem Eng
DOI: 10.1016/j.jece.2021.106296
sha: 08c029df82b089093322d1aeef14322fe7081892
doc_id: 697049
cord_uid: zgy1a6m5

Upon the outbreak of COVID-19 pandemic, detection and quantification of SARS-CoV-2 genetic material in domestic wastewater have led to an increase in the efforts to define and implement the wastewater-based epidemiology (WBE). This application provides valuable information to define local contamination monitoring, emergence of COVID-19 and its variants and many other aspects to cope with and control the pandemic. WBE surveillance, however, requires several consecutive steps such as sampling, pretreatment and concentration of samples, and detection and quantification of SARS-CoV-2 genetic material in wastewater. In this review paper, the literature regarding to all these applications reviewed considering their advantages, disadvantages as well as their applicability. A specific emphasis was placed on the last step, detection and quantification since it covers the most critical procedure for concentrating the virus before measurement. Evaluation of the existing data indicating ultrafiltration, polyethylene glycol (PEG) precipitation and electronegative membrane filtration (ENMF) were the most promising techniques for concentration. The ongoing studies are proposed to be continued within the context of standard methods. Future research needs are delineated and suggestions are made for details.

Wastewater surveillance approach was first introduced to track pharmaceuticals and personal care products in the aquatic environment as well as illicit drug use in a community in 2001 [1] . During the last two decades, this approach has been used to predict initial concentration of pharmaceuticals by means of the analysis of their pharmaceutically active substances or metabolites excreted by humans in wastewater since those substances have persistence against degradation to some degree and cause problems in the environment. This approach has also been adapted to monitoring and assessment of the occurrence and re-emergence of pathogenic viruses such as poliovirus, hepatitis virus in wastewater as an indicator of pathogenic diseases and called a wastewater-based epidemiology (WBE) surveillance. WBE surveillance has been established as a tool to detect poliovirus during the global eradication programme since 2003 [2] .

Owing to its well-known success in environmental surveillance for enterovirus infections, the global water sector has mobilized to search and utilize the WBE for SARS-CoV-2 surveillance [3] . At the early stage of ongoing COVID-19 pandemic, scientific data have proved that genetic material of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) should be present and can survive in wastewater as well as in wastewater treatment associated sludge. Therefore, WBE surveillance of SARS-CoV-2 genetic signal in sewersheds has been recommended as a promising and applicable tool to track COVID-19 pandemic by the working groups joined to International Water Research Summit held on April, 2020 [3] .

According to remarks of the summit participants, WBE SARS-CoV-2 surveillance data could provide information on potential applications summarized in Table 1 . Table 1 . Similarly, Christian Daughton who was pioneer of the WBE concept [1] defined the potential applications of WBE surveillance for assessment and management of COVID-19 pandemic by (i) a qualitative, (ii) a semi-quantitative, and (iii) a quantitative approach [4, 5, 6, 7] .

Qualitative approach is used to assess whether SARS-CoV-2 outbreak is present or not. Semiquantitative approach is utilized to monitor acceleration or deceleration of infection within an individual community. Quantitative approach reveals nationwide status and trends of outbreak due to intercomparison of infection levels across communities [5] .

For all aforementioned potential applications, the heart of WBE surveillance is the detection and quantification of SARS-CoV-2 genetic material in wastewater via standard protocols. In these protocols, sampling guidelines and detection and quantification methods must be clearly defined. Such methods consist of several steps (Fig. 1 ).

As outlined in Figure Therefore, the present review aims to compile and evaluate the data published so far on sampling, concentration, and extraction for the detection and quantification of SARS-CoV-2 genetic material in wastewater and wastewater treatment associated sludge. In this paper "wastewater" will refer to domestic wastewater unless otherwise is stated.

At the early stage of COVID-19 pandemic, primary purpose of collection of wastewater sample was to prove the presence of SARS-CoV-2 in wastewater as well as to J o u r n a l P r e -p r o o f demonstrate the usability of WBE surveillance for the analysis trends/changes in occurrence.

Considering the fact that composite sample is more homogenous and representative than grab samples, this type of sample was collected from influent of wastewater treatment plants (WWTPs) for these purposes. Duration of the composite samples were ranged from 4 to 24

hours. Some composite samples were prepared as a volume-weighted mixture of a few grab samples. Considering composite samples can inform only average concentration of the SARS-CoV-2 genetic material in wastewater during the sample collection period while grab samples allow monitoring the variation of SARS-CoV-2 signals in sewage throughout a day as well as for specific time-periods, snapshot samples were also taken from influent flow of WWTPs ( Fig. 2) . Some grab samples were also withdrawn from pumping stations and manholes located near hospitals, university or retirement home residents to find out a relation between the detected SARS-CoV-2 gene material and the clinically confirmed cases. The fate of SARS-CoV-2 virus through WWTP was monitored using sewage sludge samples taken from different units as grab samples representing snapshots.

Both composite and grab samples used in the studies are defined in Table 2 and 3 that will be given and discussed in the forthcoming sections. As seen in Fig. 2 [10] .

Earlier data indicated that SARS-coronavirus (SARS-CoV) isolated from the sputum of a patient could be thermally inactivated and incubation at 60 C for 30 min provided complete elimination of SARS-CoV [12] . Therefore, SARS-CoV-2 virus in wastewater or wastewater treatment sludge samples were thermally inactivated by pasteurization on water bath at 60 C for 90 min to ensure the safety of laboratory personnel and the environment [11, 13, 14, 15, 16] .

As detailed in Tables 2 and 3 given in the next section, some detection and quantification analyses were initiated by centrifugation and/or filtration to discard bacterial debris and coarse particles in wastewater. Centrifugation speed ranged from 1840 g to 10000

g for a timespan varying between 5 and 45 min [17] . In a few studies, filtration through either 0.22 m polyester sulfone membrane [11, 15, 18] or 100 m filter [19] was applied as a first [20] step to remove coarse particles. Some concentration applications based on precipitation were also performed without centrifugation or filtration step [21, 22, 23, 24] . It should be noted that the discarding bacterial debris and coarse particles in the sample by a separation technique such as centrifugation and/or filtration may cause negative interference since the partition of SARS-CoV-2 virus into the coarse particle is neglected. To the best of our knowledge, until now just a few studies have paid attention to this negative interference [8, 25, 26, 27] . In a study, WWTP sludge samples were agitated at 100 rpm for 30 min before centrifugation step, so as to release SARS-CoV-2 virus into the aqueous phase [27] . The contribution of partition into solids separated was determined by analysis of this fraction in studies of Kitamura et al [8] , D'Aoust et al. [25] and Westhaus et al. [26] . The data indicated that the loss of SARS-CoV-2 genetic material by solid separation should not be disregarded as discussed in the following section.

J o u r n a l P r e -p r o o f

Upon entering the sewer systems via human excretions i.e. stool and urine, SARS-CoV-2 genome becomes diluted under the detection limit [28] . Therefore, primary concentration of SARS-CoV-2 genetic material in wastewater is an essential step prior to RNA extraction to obtain more quantitatively accurate and consistent results. Cryptosporidium and Giardia in water [29] . Nevertheless, at the early stage of COVID-19

pandemic, no data existed on the potential use of these concentration techniques in the detection and quantification of SARS-CoV-2. Therefore, intensive and remarkable efforts have been made to tailor these techniques for the concentration of enveloped viruses particularly SARS-CoV-2 from wastewater within the context of WBE SARS-CoV-2

surveillance.

RNA extraction is another important step following viral concentration. In this step, RNA is isolated and purified without damaging from the concentrated sample. Organic extraction, silica-membrane based spin column technology, or paramagnetic particle technology has been commonly employed for this purpose [30] . Organic extraction may often create RNA contamination with proteins, other cellular materials, organic solvents such as phenol-chloroform, or ethanol [30] . Both silica-membrane spin column and paramagnetic particle technologies provide total intact RNA with low levels of contamination from proteins and other cellular materials while they cause significant levels of genomic DNA contamination [30] . Automated extraction kits based on these three techniques are also commercially available. Recently a list of commercial kits to be confidently used for SARS- qPCR in a few studies [25, 33, 34] . Table 2 and 3 summarize molecular techniques used in the studies together with primers/probes targeting different specific SARS-CoV-2 genes.

In the forthcoming subsections, the data published so far from all over the world have been compiled, discussed and evaluated by classifying them into four main groups, namely ultrafiltration, PEG precipitation; electronegative membrane filtration; skimmed milk or aluminium driven flocculation.

To the best of our knowledge, until now ultrafiltration (UF) based on size exclusion has been the most tested technique for concentration of SARS-CoV-2 genetic material from wastewater [8, 10, 15, 18, 19, 26, 27, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46] . In these studies, UF applications were performed using low volume of pre-centrifuged and/or filtrated wastewaters varied between 40 and 400 mL at centrifugation speeds between 3000 g and 4000 g. In these applications, centrifugal filters with different nominal molecular weight limit (NMWL) ranging from 10 to 150 kDa were utilized [17] .

Ahmed and co-workers [36] performed a study to validate the RT-qPCR assay PMMoV improved by double UF application as they reduced the level of the concentrate to be used for RNA extraction. BCoV was recovered by CeUF from the pre-centrifuged solid extraction, the partition of BCoV to solids in wastewater was concluded to be insignificant. Westhaus et al. [26] investigated the partition of SARS-CoV-2 genetic material into the solids in the wastewater. [50] . A correlation between the detected gene copies and the number of clinically reported Covid-19 cases was evident in their study. A similar correlation was also pronounced in the studies of Martin et al.

[39], Gerrity et al. [19] and Trottier [47] . Also, Baldovin et al [10] was reported the WBE detection power as at least equal to 1 clinically confirmed case out of 388 to 822 inhabitants based on their hospitalization data and their SARS-CoV-2 genetic material detections obtained from RT-qPCR assays targeting genes ORF1b-nsp14 and N.

Evaluation of above mentioned data indicated that UF, particularly double UF, yielded more sensitive results than other concentration techniques comparatively tested. It could be modified as a standard method owing to its consistence performance, satisfactory recovery rate, no need of eluting [51] , and very short operation time (max 1 hour) [52] . Together with this promising capability of recovery SARS-CoV-2 from wastewater, it should be kept in mind some disadvantages such as requirements of back-flushing, addition of blocking solution such as glycine or beef extract, and immobilization [51] .

Polyethylene glycol (PEG) precipitation is an aqueous polymer two-phase separation based on liquid-liquid partition ( [53] . This concentration method and its slightly modified applications such as overnight incubation [27, 34, 46, 54] or dextran addition [13, 14] have implemented to enrich SARS-CoV-2 genetic material in wastewater as well as treatment sludge at pH values of 6.5-7.5. Figure 3 schematically illustrates the main steps of PEG precipitation adapted and applied for the recovery of SARS-CoV-2 genome from wastewater in the recent researches. As can be seen in Table 3 , PEG precipitation is the second most applied technique to concentrate SARS-CoV-2 genetic material from wastewater [8, 11, 13, 14, 15, 16, 18, 21, 24, 25, 27, 34, 40, 41, 46, 48, 54, 55, 56, 57 ].

Insert Figure 3 . California wastewater treatment plants (WWTPs) in May 2020 [16] . The viral ranged from [58] flocculation as described in the study of Randazzo et al [22] . Their results showed that both methods were efficient to recovery SARS-CoV-2 from wastewater matrix, but PEG precipitation exhibited higher limit detection (4.3×10 6 gc L -1 ) than PAC flocculation (4.3×10 5 gc L -1 ). Hasan et al. [15] concentrated SARS-CoV-2 RNA using the protocol of Wu et al [11] for detection in the wastewater samples. The sampling program was planned to monitor Pseudomonas phage φ6 recovery to SARS-CoV-2 was searched [40] . In the study, These rates were higher than those of PEG precipitation (SARS-CoV-2/RdRp-IP4 (23.5 ± 15.0%) and SARS-CoV-2/E (45.0 ± 44.6%)). Based on their findings they deduced that overall performance of the ultrafiltration using the Centricon® 70-Plus 100 kD device coupled with RNA extraction performed phenol-chloroformisoamyl alcohol purification approach was superior to that of the PEG precipitation.

A comparative study for PEG precipitation and alum flocculation methods was made by Pérez-Cataluña et al [54] using an urban wastewater sample seeded with gamma-irradiated SARS-CoV-2, porcine epidemic diarrhea virus (PEDV), and mengovirus (MgV). Mean quantitation level for SARS-CoV-2 at 95% and 50% confidence intervals was determined as 1.7 genome equivalent (ge) mL -1 by tenfold serial dilution of the seeded sample of 200 mL.

Mean recovery efficiencies were determined as 52.8 ± 18.2% and 11.1 ± 4.9% for SARS-CoV-2 and MgV, respectively for PEG precipitation. Alum flocculation yielded similar but lower recovery efficiencies than those of PEG precipitation for SARS-CoV-2 (max 30. 

Virus adsorption-elution (VIRADEL) utilizes either electronegative or electropositive membrane filters to concentrate viruses from wastewater. Separation of virus particles is realized by attachment onto a cellulose nitrate membrane filter with pore size 0.45m via saltbridging which is sometimes supported by addition of MgCl 2 or NaCl during electronegative membrane filtration (ENMF) [53] .

Ahmed et al. [43] applied ENMF as a concentration technique to a raw wastewater with/without different pre-conditioning applications namely acidified (2 N HCl; pH 4) and found to be more efficient method than M2 (1.3 × 10 4 cg L -1 ). As a result, electronegative membrane-vortex followed by RNA extraction using the QIAamp Viral RNA Mini Kit was suggested as an applicable concentration method for WBE SARS-CoV-2 surveillance.

Similarly, ENMF using nitrocellulose membrane with pore size of 0.45-μm was determined to be more efficient concentration method than UF using a centrifugal filters with a cut-off of 10

KDa at 2200 g for 20 min [66] . In this study, grab samples were taken from after coarse and fine screening units (influent), activated sludge recirculation (secondary sludge) systems and after the disinfection units (effluent) of two WWTPs located in Mexico. Molecular assays were performed using RT-qPCR assays targeting the S protein and N protein. SARS-CoV-2 RNA level of secondary sludge was higher than those of influent samples and SARS-CoV-2 was negative in all effluent samples. These data confirmed the importance of solid-liquid interaction of SARS-CoV-2 in wastewater.

Electropositive NanoCeram column filtration was applied to wastewater samples collected from a WWTP located in southeast Michigan, USA [67] . SARS-CoV-2 N1 genes were quantified using a two-step quantitative RT-qPCR. SARS-CoV-2 N1 was detected in all wastewater samples (54/54) and quantified as in the range of 10 4 -10 5 gc L -1 .

Based on limited data, it could be deduced that preconditioning applications such as addition to MgCl 2 might improve the SARS-CoV-2 recovery efficiency of ENMF and electropositive filters were also capable of concentrating SARS-CoV-2 genetic material from wastewater. Hence, further studies are needed to assess and maximize the effectiveness of both concentration techniques.

During the skimmed-milk flocculation (SMF) application, viruses eluted with glycine alkaline buffer are flocculated with skimmed milk at pH 3.5. This concentration technique has been adapted to wastewater by Calgua et al. [68] . Fig. 4 shows Calgua's method applied to the concentration of SARS-CoV-2 genetic material from wastewater [20, 21, 23] .

To the best of our knowledge, until now only a few researches have been realized using SMF to concentrate SARS-CoV-2 from wastewater. As mentioned in the previous subsection, SMF was one of the concentration methods tested by Barril Aluminium-driven flocculation produces fresh Al(OH) 3 flocs onto which viruses are adsorbed at pH 6.0. Only one research group has employed this concentration technique to recover SARS-CoV-2 genetic material from wastewater. Figure 5 shows the steps followed by this group ( [22, 58] . The first study was performed using the influent and effluent samples taken from three WWTPs located in Valencia [58] , the second was accomplished using the samples collected from 6 WWTPs in the Region of Murcia (Spain) characterizing a low prevalence area in Spain [22] . Porcine Epidemic Diarrhea Virus (PEDV) and MgV were used in the method validation. For the influent samples PEDV and MgV recovery efficiencies were determined as 10 ± 3.5% and 10 ± 2.1%, respectively [22] . SARS-CoV-2 N1, N2 and N3 were quantified as 5.1 ± 0.3, 5.5 ± 0.2, and 5.5 ± 0.3 log 10 gcL -1 , respectively in the Valencia's samples.

Existing data indicated that skimmed milk flocculation and aluminium driven flocculation used for wastewaters and contaminated receiving waters may also be considered as promising alternatives if further studies support their high performance maybe with some modifications. Comparative studies should be performed in order to clarify these critical points for general use listed in Table 1 . Findings have already proved applicability of present concentration techniques for recovery of SARS-CoV-2 genetic material. Among the present techniques, UF, PEG precipitation and ENMF exhibit higher potential to be improved as standard methods.

Their variability, reproducibility, and reliability of these concentration methods should be evaluated by intra and inter laboratory calibration studies in order to be approved as standard methods. Some of the methods have been shown to have the potential of being used as a measure of SARS-CoV-2 removal performance of the domestic wastewater treatment units.

The studies in the field should be continued within the context of "standard methods", so the unified procedures could be derived. As indicated in the literature evaluation some preconditioning applications may significantly enhance the sensitivity or detection level of the concentration methods. Therefore, the further researches are also expected to account for preconditioning processes and their basis of the application. In parallel with the development of the methods, comparative studies would be quite useful to assess the strength and weakness of the individual method against the properties of raw and treated wastewaters as well as treatment sludge. The literature reviewed in this paper did not specify the wastewater strengths except for a few studies. Therefore, a relation between SARS-CoV-2 genetic material and wastewater characteristics could not be established. Similar calibration studies should be done for both RNA extraction techniques and modern molecular assays. Declaration of interests ☒ 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. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Prof. Dr. Işık Kabdaşlı

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The authors extend sincere gratitude to all scientists, researchers, laboratory teams and other employees for their efforts who involved in the studies in this field. They also wish to acknowledge İstanbul Technical University for the acquisition of the latest literature regarding the COVID-19 pandemic used in this review.