A Simple Method to Detect SARS-CoV-2 in Wastewater at Low Virus Concentration

Background Since its initial appearance in December 2019, coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread globally. Wastewater surveillance has been demonstrated as capable of identifying infection clusters early. The purpose of this study was to investigate a quick and simple method to detect SARS-CoV-2 in wastewater in Thailand during the early stages of the second outbreak wave when the prevalence of the disease and the virus concentration in wastewater were low. Methods Wastewater samples were collected from a hospital caring for patients with COVID-19 and from 35 markets, two of which were associated with recently reported COVID-19 cases. Then, samples were concentrated by membrane filtering prior to SARS-CoV-2 detection by RT-qPCR. Results SARS-CoV-2 RNA was detected in the wastewater samples from the hospital; the Ct values for the N, ORF1ab, and S genes progressively increased as the number of patients admitted to the treatment floor decreased. Notably, the ORF1ab and S genes were still detectable in wastewater even when only one patient with COVID-19 remained at the hospital. SARS-CoV-2 RNA was detected in the wastewater samples from fresh market where COVID-19 cases were reported. Conclusions Our findings suggest that wastewater surveillance for SARS-CoV-2 is sensitive and can detect the virus even in places with a high ambient temperature and relatively low prevalence of COVID-19.


Background
Wastewater surveillance for SARS-CoV-2 has been demonstrated to be a feasible and sensitive method for assessing the prevalence and monitoring the transmission of SARS-CoV-2 in various countries and situations during the ongoing COVID-19 pandemic. It was initially shown that SARS-CoV-2 can be detected in both untreated [1][2][3][4][5] and treated wastewater [6] in several countries. Similar results from additional studies in multiple geographical areas have led to the United States Center for Disease Control to recommend wastewater surveillance for SARS-CoV-2 [7]. e spread of COVID-19 appears to be lessened by warm and wet weather [3], and there is a negative correlation between temperature and COVID-19 prevalence across countries. Notably, moderate temperature increases to 34°C can disrupt the structure of SARS-CoV-2, while humidity has very little impact [8]. For the related virus, severe acute respiratory syndrome (SARS), UV irradiation of >90 μw/ cm 3 to the culture medium for 60 min can completely impair viral infectivity [9]. Furthermore, UV radiation has been shown to have a significant association with the incidence of COVID-19, which may help to flatten the epidemic [10]. To date, most studies examining the utilization of wastewater surveillance for SARS-CoV-2 were performed in countries with cold weather and/or a high COVID-19 prevalence. For tropical countries, at least in India during 2020, when the average temperature was 25.78 degrees Celsius (°C) [11], SARS-CoV-2 could be molecularly detected in influent wastewater from a water treatment plant. However, at the time of that study, the prevalence of SARS-CoV-2 in India was very high [12]. ailand is located in the tropical area where the climate is warm to hot year-round with average temperatures during winter, summer, and rainy season at 26.2°C, 29.7°C, and 28.2°C, respectively [13]. Overall, the prevalence of SARS-CoV-2 infection has been relatively low compared to other countries, particularly during the earlier waves of the pandemic. To help facilitate wastewater surveillance in low prevalence areas, we developed and examined in the present study a quick and simple method for detecting SARS-CoV-2 in wastewater in ailand, where the ambient temperature is high, and the prevalence of reported COVID-19 cases is relatively low during the early stages of the second outbreak wave.

Materials and Methods
e study was approved by the Institute Biosafety Committee (Protocol ID: MU 2021-002), and wastewater samples potentially containing SARS-CoV-2 were properly processed in accordance with standard biosafety guidelines from the World Health Organization [14].

Sample Collection.
During January and February 2021, at the time of the second COVID-19 outbreak in ailand, grab samples of wastewater were obtained from Chakri Naruebodindra Medical Institute (CNMI), a hospital treating patients with COVID-19 in Samut Prakan province, ailand. All wastewater samples were collected downstream to the isolation ward for patients with confirmed COVID-19 and before the influent to a water treatment station. All samples were transported on ice to the laboratory for processing.
During February 2021, after social distancing and the closure of schools and specific crowded areas were implemented during the second wave of the SARS-CoV-2 pandemic in ailand of which the epicenter was Samut Sakhon province (45 km southwest of Bangkok), sewage wastewater samples were collected from 46 open fresh markets in the Bangkok metropolitan area and in the anyaburi district of Pathum ani province. e first confirmed COVID-19 case in Pathum ani province, part of outer northern Bangkok, was found at the Pornpat market and officially reported on 22 December 2020. In the period from 4 to 13 January 2021, the SARS-CoV-2 infection rate rose to 1 Pornpat market wastewater samples (P1-P4) were collected from the sewage pipeline network around the four corners of the market. Pornpat market samples were also collected from two water sampling sites (P50P6) in the Rangsit canal to check for contamination and from another two sites (P7-P8) at sewage pipelines from dwellings next to the Pornpat market. For the Suchart market, two wastewater samples (S1-S2) were collected from the sewage pipeline in the market, and another sample (S3) was collected from the Rangsit canal. For the Rangsit market, three waste water samples (R1-R3) were collected from the sewage pipeline network that passed through the middle of the market (Supplement Figure S1).
Grab samples of wastewater (500 mL/sample) were collected in clean plastic bottles from the wastewater drainage/sewage management system associated with each market and transported on ice to the laboratory, where they were stored at 4°C until use in further analysis.

Sample Preparation and Concentration.
e sample preparation and concentrating method was adapted from the work of Ahmed et al. [15]. A subsample (100-400 mL) of each collected grab sample of wastewater was centrifuged at 3000 ×g for 10 min at room temperature to separate out the sediment. e resulting supernatant was then filtered by using a mixed cellulose ester membrane filter (pore size, 0.45 µm; diameter, 47 mm; GE Healthcare, Chicago, IL, USA) attached to a disposable Millicup ™ -FLEX filtration unit (Merck Ltd, Darmstadt, Germany), followed by applying the vacuum pump system to the assembly filtration unit until filtration was complete. Subsequently, the membrane filter was removed and placed in a sterile 5 mL tube. DNA/RNA Shield ™ (1 mL) and 0.1 g of ZR BashingBead (Zymo Research, Sigma, Irvine, CA, USA) were added to each tube, after which the tubes were stored at −80°C until use in further RNA isolation.

RNA Extraction.
To elute the viral RNA from the filtered mixed cellulose ester membrane, the preparation solution was first mixed 10 times (60 s each) with a vortex mixer at near maximum speed. After being mixed, 400 µL of the solution was transferred into a new nuclease-free tube. e viral RNA was then extracted by using the viral RNA mini kit (Qiagen, Hilden, Germany) in accordance with the manufacturer's protocol. e concentration and purity of the extracted RNA were determined by using a Nanodrop ™ ( ermo Fisher Scientific, Waltham, MA, USA). e absorbance readings at 260 nm and 280 nm (260/A280 ratios) are commonly used to determine the purity of nucleic acid. A general acceptable range for 260/280 ratios is 1.9-2.1. control ( ermo Fisher Scientific) was used as a positive control, MS2 Phage control was used as an internal positive control, and DNase/RNase-free water was used as a negative control. Each RT-qPCR experiment was performed on a ViiA 7 Real-Time PCR instrument (Applied Biosystems, Waltham, MA, USA) using the following thermocycling conditions: 2 min at 25°C for UNG incubation to eliminate amplicon carryover, 15 min at 50°C for reverse transcription, 2 min at 95°C for predenaturation, and 40 cycles of 3 s at 95°C and 30 s at 60°C for denaturation, annealing, and extension. After every amplification cycle, the fluorescence intensity was measured at 60°C. Results were classified as positive for SARS-CoV-2 detection if they included positive results, defined as cycle threshold (Ct) < 37, for two or more SARS-CoV-2 target genes. An individual assay result of 37 ≤ Ct ≤ 40 was considered to be inconclusive; samples with inconclusive results were repeated.

SARS-CoV-2 Detection and Quantification. For SARS-
A 10-fold serial dilution of TaqPath ™ COVID-19 control amplification was performed to generate the standard curve used for SARS-CoV-2 quantification.

Sensitivity of SARS-CoV-2 Detection in Wastewater.
To determine the lower limit of detection of the assay kit used in this study, we established a standard curve with 10fold serial dilutions of 2019-nCoV DNA control from the RT-qPCR Kit, ranging from 1×10 4 -1 copies/µL. An inverse linear relationship was generated against each of the three target genes. e mean Ct values ranging from 26.9 ± 0.1 to 35.9 ± 1.1 for the ORF1ab gene, 26.7 ± 0.1 to 33.6 ± 0.4 for the N gene, and 26.1 ± 0.1 to 32.9 ± 0.6 for the S gene corresponded to concentrations of 10 4 -10 2 copies/µL (Supplement Table S1). For the sensitivity of the used method on grab wastewater, left-over grab wastewater samples negative for SARS-CoV-2 viral RNA stored at -80°c were pooled and mixed for analysis in a spike study. Seven hundred mL of the pooled samples was divided into 14 equal aliquots of 50 mL and processed in duplicate. Two aliquots of no-spike wastewater were autoclaved, to further ensure the absence of SARS-CoV-2 RNA in the samples, and were used as blank samples. Ten aliquots were individually spiked with 10-fold serial dilutions of inactivated culture medium of SARS-CoV-2 with 10% TRIzol ™ reagent (cat. no. 15596026, Life Technologies Corporation, Carlsbad, CA 92008). Genomic copies (GC) number in all samples was determined by RT-qPCR. Each 50 mL spiked wastewater aliquot was then concentrated by the method adapted from the work of Ahmed et al. [15] before further RNA extraction and RT-qPCR as described in Materials and Methods. Table 1 shows the results of the spike experiments with serially diluted virus culture medium samples into pooled wastewater samples negative for the virus. SARS-CoV-2 could be detected in 15/15 in all seeded samples with Ct values ranging from 18.28 ± 0.16 to 28.60 ± 0.41 for the N gene, 18.94 ± 0.06 to 29.13 ± 0.85 for the ORF1ab gene, and 19.37 ± 0.11 to 29.77 ± 1.54 for the S gene, respectively. Overall, the assay was sensitive enough to detect SARS-CoV-2 down to 1.63 ± 0.47, 1.20 ± 0.49, and 1.20 ± 1.51 copies/mL for the N, ORF1ab, and S genes, respectively. No virus was detected from all 4 samples extracted from no-spike pooled wastewater with or without being autoclaved before the extraction.

Sensitivity of SARS-CoV-2 Detection in Wastewater.
As shown in Table 2, the Ct values for the N, ORF1ab, and S genes progressively increased as the number of patients admitted to the treatment floor decreased. Notably, two out of the three SARS-CoV-2 genes were still detectable in wastewater even when only one patient was present in the COVID-19 isolation ward. e ORF1ab and S genes appeared to be more sensitive for detecting the presence of patients with COVID-19 from wastewater. As the number of patients with COVID-19 decreased, the Ct values for both these genes increased. ere was a significant correlation between the number of cases and the Ct values of the ORF1ab gene (r � −0.99, p < 0.05), whereas such correlation for the S gene tended to reach statistical significance (r � −0.98, p � 0.06). For the S genes, there were only 2 detectable samples, and the statistical analysis was not performed.

SARS-CoV-2 Surveillance in Wastewater from Fresh
Markets in Bangkok. Wastewater samples were collected from 46 open fresh markets in the Bangkok metropolitan area. e daily number of reported cases in Bangkok during this period is shown in Figure 1, and a map of the markets from which the wastewater samples were collected is in Supplement Figure S2. All samples collected and tested during this period were found to be negative for SARS-CoV-2. ere was no major outbreak of SARS-CoV-2 infection in Bangkok for up to 1 month after the wastewater collection period.

SARS-CoV-2 Surveillance of Wastewater from Fresh Markets in Pathum ani Province.
e SARS-CoV-2 RNA detection results for these samples are presented in Table 3. SARS-CoV-2 RNA was detected in three of the four samples (P1-P2, P4) from the Pornpat market sewage pipeline network, but the fourth sample (P3) produced an inconclusive result. Only the ORF1ab gene was detected in all four samples from the Pornpat market sewage pipeline network (P1-P4); similarly, this gene was also the only one detected in both of the sewage pipeline samples from the Suchart market (S1-S2). Furthermore, SARS-CoV-2 RNA was detected in the samples from sewage pipelines from dwellings next to the Pornpat market (P7-P8). In contrast, SARS-CoV-2 genetic material was not detected in any samples from the Rangsit canal (P4-P5, S3) or the Rangsit market (R1-R3).

Discussion
In the present study, we demonstrated the performance of a quick and simple method for detecting SARS-CoV-2 in wastewater in Bangkok and the surrounding areas despite the high ambient temperature and low prevalence of COVID-19 in this location. Previous studies, such as those performed in the Netherlands [2] and Australia [1], have applied various methods of filtering, concentrating, and PCR to detect SARS-CoV-2 in sewage samples. Here, we chose to use membrane filtering and concentrating, which have been demonstrated as reliable methods for detecting SARS-CoV-2.
Recent studies in many countries identified SAR-CoV2 viral RNA in wastewater, sewage sludge, and river water Table 1: SARS-CoV-2 detection from pooled wastewater negative for SARS-CoV-2 with and without SARS-CoV-2 spike.

SARS-CoV-2 seeded (GC)
N ORF1ab S Mean Ct + SD Copies/mL ± SD Mean Ct + SD Copies/mL ± SD Mean Ct + SD Copies/mL ± SD 2 × 10e4 18.28 ± 0.16 4,821.14 + 1,250. 18    using two or more genetic parts of the viral genome [17]. ree different SARS-CoV-2 genomic regions, Orf1ab gene, N gene, and S gene, were used to detect virus RNA in wastewater in Chile [18], India [12,19], and Germany [20]. e studies revealed that the genome copy number of the viruses increased progressively, corresponding to an increase in the estimated number of virus infections in the community and the affected area. Furthermore, partly similar to our study, the Ct values have been demonstrated to negatively correlate to the effective reproduction number (Rt), daily COVID-19 hospitalization with a 33-day time delay, and daily changes in percent positivity among tested samples [21].
As for now, there are no assay targets recommended for identifying SARS-CoV-2 in wastewater. Several SARS-CoV-2 gene targets were commonly used for RT-qPCR detection such as N, E, S, RNA-dependent RNA-polymerase (RdRp, also known as nsp12), and open reading ORF1ab [22]. Bivins et al. reviewed the SARS-CoV2 RNA wastewater surveillance about the variability of basic and essential information for reverse transcription-quantitative PCR (RT-qPCR) assay parameters such as SARS-CoV-2 gene target, the standard curve parameters of y-intercept, slope and/or efficiency, and r2 value. ey screened 208 RT-qPCR assays from 46 preprint and 36 peer-reviewed publications and found the assays targeting 130 N gene, 25 targeted ORF1, 23 targeted the E gene, 19 targeted RdRp, and 10 targeted the S gene, whilst one did not report any target molecule. Quantification SARS-CoV-2 RNA in wastewater targeting the US CDC N1 and N2 accounted for 45% of the RT-qPCR assays reported and N1 was tested more frequently (39%) than the US CDC N2 (32%) [23]. erefore, we examined three different genes of SARS-CoV-2, ORF1ab, N protein genes and S protein genes, using the commercial PCR test kits, TaqMan ™ 2019-nCoV Assay Kit v2 (Life Technologies Corporation, USA) for SARS-CoV-2 diagnosis of the virus genome in wastewater sample. is assay kit has been approved for marketing in ailand by the Food and Drug Administration (FDA) ailand under an evaluated for emergency use authorization (EUA) since June 4th, 2020 [24].
is study protocol allowed SARS-CoV-2 to be detected even when the number of apparently infected individuals in the wastewater catchment area was as low as one. Interestingly, although the RT-qPCR Ct values were related to both the absolute number and the percentage of persons infected with SARS-CoV-2 in the catchment area, the Ct values were more closely related to the absolute number of infected persons. is result is likely a consequence of the high sensitivity of RT-qPCR, which can detect SARS-CoV-2 RNA in samples with as little as 21 copies per reaction for the N1 gene [25]. Our findings also suggest that, at the building level, the amount of daily wastewater production may not only be related to the number of people using the sewage system. Importantly, our study confirms that SARS-CoV-2 can be detected in wastewater from a number of areas in ailand, where the ambient temperature is high and there is abundant sunshine all year round and the average temperature obtained from the ai Meteorological Department [13] during collection of samples is 25.7 and 28.4 for January and February 2021, respectively. e average daily maximum UV index is 10 in January and up to 12 in February.
Our finding is in line with a report from India, where the weather is relatively similar to that of ailand; SARS-CoV-2 could be detected in wastewater from India during the early phase of the SARS-CoV-2 pandemic [12]. In that study, water was sampled from a water treatment plant in Ahmedabad, for which the catchment areas included a hospital that was treating patients with COVID-19. Ahmedabad is located close to the equator and has a high ambient temperature, averaging 27.1°C [26]. e number of COVID-19 cases in Ahmedabad during the course of that study was approximately 5,000-10,000, which is much higher than the COVID-19 prevalence in ailand during the present study. A few other countries in Asia have also reported the successful detection of SARS-CoV-2 in wastewater. In Japan, during March to May 2020, wastewater samples were collected from several water treatment plants, and the SARS-CoV-2 detection frequency was found Journal of Environmental and Public Health to increase with the number of reported COVID-19 cases [27]. Interestingly, SARS-CoV-2 could be detected even when the number of COVID-19 cases was <1.0 per 100,000 people. Another study with sample sources and a setting similar to ours identified SARS-CoV-2 in wastewater from a hospital in China [28], although unlike our study, the number of COVID-19 cases in China at the time of that study was very high. Our study is in line with a report from Wannigama et al. [29], who reported the detectability of SARS-CoV-2 in wastewater in Bangkok and suggested that wastewater can be used as a complementary source for detecting the viral RNA and predicting upcoming outbreaks. ey monitor SARS-CoV-2 RNA in wastewater prior to the second outbreak in ailand during the rainy season and winter.
In the present study, RT-qPCR performed using a  [30]. Using the RNA-dependent RNA polymerase (RdRP), N, and S genes as targets, another study on wastewater samples found that the amplification efficiencies were 93%, 87%, and 84%, respectively [31]. e sensitivity differences among gene targets and different studies have been suggested to reflect differences in the abundance of SARS-CoV-2 in wastewater according to the community COVID-19 pandemic level and the methodologies applied for viral RNA detection, including those for virus concentration, RNA extraction, and RT-qPCR assay [32].

Conclusions
(i) e membrane filtering-based method described here is a rapid, extremely simple, and sensitive approach for the detection of SARS-CoV-2 in wastewater from areas with low numbers of COVID-19 cases (ii) Wastewater monitoring for SARS-CoV-2 is sensitive and can detect the virus even in places with a high ambient temperature and relatively low prevalence of COVID-19 (iii) is data is useful for community SARS-CoV-2 surveillance and prevention of the spread of coronavirus disease or COVID-19 Data Availability e data used to support the finding of this study are included within the article.
Ethical Approval e study was approved by the Institute Biosafety Committee (protocol ID: MU 2021-002), and wastewater samples potentially containing SARS-CoV-2 were properly processed in accordance with standard biosafety guidelines from the World Health Organization.

Conflicts of Interest
e authors declare conflicts of interest. Acknowledgments is study was supported by the Ramathibodi Foundation. Table S1. Quantification of a tenfold serial dilution of 2019-nCoV DNA control by real-time qPCR for the lower limit of detection assay. Figure S1. Location of markets in Pathum ani from which wastewater samples were collected. e symbol represents sites that tested negative, the symbol represents sites that tested inconclusive, and the symbol represents sites that tested positive for SARS-CoV-2 RNA. Figure S2. Location of the markets from which wastewater samples were collected in Bangkok (Supplementary Materials)