key: cord-0877808-364ap4qj
authors: Hong, P.; Rachmadi, A. T.; Mantilla-Calderon, D.; Alkahtani, M.; Bashwari, Y. M.; Al Qarni, H.; Zhou, J.
title: How early into the outbreak can surveillance of SARS-CoV-2 in wastewater tell us?
date: 2020-08-22
journal: nan
DOI: 10.1101/2020.08.19.20177667
sha: 12ac57bf5e2646f252f0c23fc9217ae6f3f7eec8
doc_id: 877808
cord_uid: 364ap4qj

There is increasing interest to use wastewater-based surveillance of SARS-CoV-2 as an early warning of the outbreak within a community. Despite successful detection of SARS-CoV-2 in wastewaters sampled from multiple locations, there is still no clear idea on the minimal number of cases needed in a community to result in a positive detection of the virus in wastewaters. To address this knowledge gap, we sampled wastewaters from a septic tank and biological activated sludge tank located on-site of a hospital. The hospital is providing treatment for SARS-CoV-2 infected patients, with the number of hospitalized patients per day known. It was observed that > 253 positive cases out of 10,000 persons are required prior to detecting SARS-CoV-2 in wastewater. There was a weak correlation between N1 and N2 gene abundances in wastewater with the number of hospitalized cases. This correlation was however not observed for N3 gene. The occurrence frequency of SARS-CoV-2 is at least 5 times lower in the partially treated wastewater than in the septic tank. Furthermore, abundance of N1 and N3 genes in the activated sludge tank were 50 and 70% of the levels detected in septic tank, suggesting poor persistence of the SARS-CoV-2 gene fragments in wastewater.

Data from the World Health Organization suggests that approximately 80% of Covid-19 infected 45 individuals exhibit only mild symptoms or are asymptomatic 1 . In particular, asymptomatic cases are 46 concerning as these individuals cannot be detected easily by the current swab testing protocols and can 47 unknowingly transmit the virus to others. Although lockdown and curfew are very effective in slowing the 48 spread of SARS-CoV-2, such intervention measures in the long term is unsustainable, and remains as a 49 key but not the only solution to mitigating . The other solution lies in increasing our testing 50 capacity. However, to swab everyone in a country for clinical surveillance over a long term period is 51 impractical considering the amount of resources and labor hours needed. In contrast, monitoring for 52 SARS-CoV-2 directly in wastewaters over a longer duration would be an alternate method to complement 53 clinical surveillance since about 39 to 65% of infected hosts, including asymptomatic carriers, shed the 54 virus through their feces, while about 6% of patients shed it through urine 2, 3 .

By sampling for the waste stream generated from a community, we therefore have a composite 56 sample that would be suitable for wastewater-based epidemiology (WBE). WBE is an approach that 57 includes the qualitative and quantitative determination of biomarkers in raw wastewater to provide 58 information on inhabitants within that wastewater catchment area. Monitoring for SARS-CoV-2 in 59 wastewater has been demonstrated in many countries, including the Netherlands, Australia, Italy, Spain 60 and US [4] [5] [6] [7] [8] [9] . In these studies, SARS-CoV-2 was sporadically detected from the sampled wastewaters, and 61 the reported abundance of SARS-CoV-2 in wastewater ranged from 19 to 2.2 x 10 8 copies/L. In some 62 instances, the WBE approach was able to detect SARS-CoV-2 in wastewater before clinically diagnosed 63 cases were made known 10 . WBE hence demonstrates potential to serve as an early warning of 64 re(emergence) of Covid-19 in communities 7 . However, without knowing the minimal number of positive 4 can be easily determined, the number of infected individuals is difficult to obtain unless an active 72 surveillance is conducted to swab test all individuals, including asymptomatic ones, in the community. To 73 circumvent this limitation, a controlled community, for example, hospitals providing treatment to 74 patients can be used as a model. In this manner, the number of patients contributing SARS-CoV-2 to the 75 hospital wastewaters are known through daily hospitalization and discharge records. Besides determining 76 the detection sensitivity of WBE using hospital wastewaters as a study model, we further evaluate the 77 correlation between detected abundance of SARS-CoV-2 gene fragments in wastewater with the number 78 of patients. The abundance of nucleocapsid gene fragments as it moves from the underground septic 79 tank to the first stage of the wastewater treatment plant situated on-site was also evaluated.

Findings from this study will provide inference to the minimal number of SARS-CoV-2 infected cases 81 needed to generate quantitative PCR (qPCR) signals associated with the virus shed into a highly diluted To determine recovery and viral nucleic acid extraction efficiency, a known concentration of murine 91 norovirus (MNV) was spiked into untreated wastewater. MNV was spiked as surrogate because it is a 92 positive-sense single-stranded RNA virus like SARS-CoV-2, and will likely be similar in terms of RNA 93 extraction efficiency. Quantitative PCR (qPCR) for MNV was performed using forward primer (5'-94 CCGCAGGAACGCTCAGCAG-3'), reverse primer (5'-GGYTGAATGGGGACGGCCTG-3') and Taqman 95 probe (5'-FAM-ATGAGTGATGGCGCA-ZEN/IBFQ-3') (Integrated DNA Technologies, Leuven, Belgium 96 11 . A six-points standard curve was generated using a synthetic oligonucleotide (gblocks @ gene fragment,

Integrated DNA technologies, IA, USA) containing 300-350 bp DNA sequence that encompasses a 98 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted August 22, 2020 . . https://doi.org/10.1101 /2020 complementary region for which the primers and probes would anneal to. An amplification efficiency of 99 102.7% was achieved. LOD for MNV qPCR reaction was 3.89-log copies/L and 10 copy/well, 100 respectively. MNV is not anticipated to be present in municipal wastewaters and hence abundances 101 detected by means of qPCR would be due to the spiking event. from 15 April to 9 July 2020, with frequencies ranging from 3 to 5 daily samples per week (Table S1 ). The 115 lower sampling frequency during the early stages of the outbreak was due to the movement restriction 116 order imposed in the city. Samples were stored at 4 o C for not more than 1 week before they were 117 processed.

Processing of wastewater samples 120 250 to 500 mL of samples were individually concentrated for viral particles using the Millipore HA 121 membrane method with slight modifications 13 . Briefly, 2 mL of 2.5 M MgCl2 was added to every 100 mL 122 of the sample, agitated for 3 minutes and left to stand for 3 minutes. The samples were then filtered 123 through the HA membrane. The HA membrane was then filtered through with 200 mL of 0.5 mM H2SO4 to 124 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted August 22, 2020. . https://doi.org/10.1101/2020.08.19.20177667 doi: medRxiv preprint adjust the isoelectric point of viruses. The viral particles retained on the HA membrane were eluted with 125 10 mL of 1 mM NaOH into a sterile collection tube that contained 100 µL of 100X tris-EDTA buffer and 50 126 µL of 100 mM H2SO4. The eluate was concentrated with Centripep YM-50 (Millipore) to approximately 127 680 µL, and an aliquot of 140 µL was extracted for its RNA using QIAmp Viral RNA kit (ThermoFisher CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted August 22, 2020. . https://doi.org/10.1101/2020.08.19.20177667 doi: medRxiv preprint A total of 6.89-log copies/L MNV was spiked into the untreated wastewater. The overall recovery and 151 RNA extraction efficiency of murine norovirus was ca. 45%, and falls within the range reported by earlier 152 studies (ranging from 10% to 73%) albeit using different concentration procedures. For example, direct 153 ultracentrifugation of 20 mL of wastewater through a 50% sucrose phase layer recovered 12% of the 154 deactivated SARS-CoV-2 spiked into the sample 17 . Aluminium hydroxide adsorption-precipitation 155 (adjusted to pH 6) method recovered on average 11% of spiked porcine epidemic diarrhea virus and 156 mengovirus, respectively, from 200 mL wastewater 8 . Ultrafiltration of 100-200 mL using Centricon Plus-157 70 (molecular weight cut-off of 10 kDa) recovered an average 73% of the spiked F-specific RNA phages 158 but with high standard deviation of 50% 7 .

In addition, the reported differences in recovery efficiencies can be due to the varying type of viral CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted August 22, 2020. . https://doi.org/10.1101/2020.08.19.20177667 doi: medRxiv preprint volume of untreated wastewater (< 500 mL) to be filtered through without significantly lengthening the CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted August 22, 2020. . https://doi.org/10.1101/2020.08.19.20177667 doi: medRxiv preprint be dependent on the viral shedding load per patient and the shedding duration. In this study, information 204 on whether patients are exhibiting gastrointestinal distress was not provided to us due to concerns of 205 breaching patients' confidentiality. Hence it is uncertain how many percentage of the patients may be (Table 1) . This indicates a certain degree of partial treatment 218 was achieved at the sampling point of the activated sludge tank.

Likewise, this level of partial treatment resulted in a lower detection frequency of SARS-CoV-2 in 220 wastewaters sampled from the biological activated sludge tank. Only 8 out of 52 samples (15.4%) 221 collected from the biological activated sludge tank were tested positive for N genes associated with 222 SARS-CoV-2. Specifically, N1 genes were detected in 5 samples, N2 genes were detected in 4 samples, 223 and 1 positive occurrence of N3 genes among the collected samples. N1 genes were detected first before 224 N2 and N3. The average abundance of N1 and N3 genes decreased by 0.3-log (i.e., 50%) and 0.5-log 225 (i.e., 70%), respectively, from the underground septic tanks to the biological activated sludge tank.

However, there was an increase by 44% in the average abundance of N2 genes sampled from the 227 activated sludge tank compared to that in the underground septic tank. This suggest a potential 228 accumulation of N2 genes in the activated sludge tank, likely due to better persistence of N2 compared to 229 N1 and N3 genes. Most of the positive detection in the biological activated sludge tank occurred in late 230 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted August 22, 2020 . . https://doi.org/10.1101 /2020 June, and coincides after a period of high cumulative numbers of hospitalized patients. Despite the slight 231 increase of N2 genes in a small group of samples, it is unlikely that the N genes will persist in the final 232 treated wastewater since the last stage of the hospital WWTP includes a final disinfection step at a very 233 high chlorine concentration (100 mg/L) and at long contact time (2 h). An earlier study determined that 6.5 234 mg/L of free chlorine and 1.5 h of contact time was already sufficient to remove 0.5 to 18.7 x 10 3 copies/L 235 of SARS-CoV-2 viral RNA to levels below detection limits 25 .

By utilizing a confined environment (i.e., a hospital with known number of Covid-19 cases), this study 239 determined the detection sensitivity of wastewater-based epidemiology for SARS-CoV-2 to be about > 240 253 persons per 10,000 inhabitants. The correlation between nucleocapsid gene abundances of SARS-

CoV-2 and the number of infected hospitalized patients was weak, suggesting the difficulty in correlating 242 the number of cases based on the gene abundances detected in wastewaters. However, it is likely that 243 when the nucleocapsid genes were detected, there is already a substantial number of cases circulating in 244 the community that would warrant immediate intervention measures to be taken. N genes do not persist 245 well in the hospital wastewater based on the lower detection frequency in partially treated wastewater. In 246 instances where the nucleocapsid genes were detected in the partially treated wastewater, N1 and N3 247 genes had already decreased by 0.3-log and 0.5-log (i.e., ca. 50 and 70%, respectively). This is with the 248 exception of N2 genes that may persist better than N1 and N3. Future studies would be needed to 249 improve the recovery efficiency of viral particles from wastewater matrix, and to determine the decay CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted August 22, 2020 . . https://doi.org/10.1101 /2020 

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted August 22, 2020 . . https://doi.org/10.1101 /2020 

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted August 22, 2020 . . https://doi.org/10.1101 /2020 

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted August 22, 2020. probe information. In 2020. 319 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted August 22, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted August 22, 2020. . https://doi.org/10.1101/2020.08.19.20177667 doi: medRxiv preprint TOC   Table S1 . Information on the sampling dates at the respective sampling point……………………………..1 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted August 22, 2020. . https://doi.org/10.1101/2020.08.19.20177667 doi: medRxiv preprint Table S1 . Information on the sampling dates at the respective sampling point. Gray cell indicates sampling was performed at the septic tank on that particular date. Green cell indicates sampling was performed at the biological tank on that particular date. Values in each cell refer to the filtered volume.

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Thuwal 23955-6900, Saudi Arabia 2

General Directorate of Environment Health, Ministry of Health

c.. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)The copyright holder for this preprint this version posted August 22, 2020 . . https://doi.org/10.1101 /2020 How early into the outbreak can surveillance of SARS-CoV-2 in wastewater tell us?