key: cord-0898335-unlmqvue
authors: Wong, Judith Chui Ching; Tan, Joanna; Lim, Ying Xian; Arivalan, Sathish; Hapuarachchi, Hapuarachchige Chanditha; Mailepessov, Diyar; Griffiths, Jane; Jayarajah, Praveena; Xiang, Setoh Yin; Tien, Wei Ping; Low, Swee Ling; Koo, Carmen; Yenamandra, Surya Pavan; Kong, Marcella; Lee, Vernon Jian Ming; Ching, Ng Lee
title: Non-intrusive wastewater surveillance for monitoring of a residential building for COVID-19 cases
date: 2021-04-29
journal: Sci Total Environ
DOI: 10.1016/j.scitotenv.2021.147419
sha: 1e90a7dab98dbafa8a8b4ea217066e4512b03c78
doc_id: 898335
cord_uid: unlmqvue

Wastewater-based surveillance for SARS-CoV-2 has been used for the early warning of transmission or objective trending of the population-level disease prevalence. Here, we describe a new use-case of conducting targeted wastewater surveillance to complement clinical testing for case identification in a small community at risk of COVID-19 transmission. On 2 July 2020, a cluster of COVID-19 cases in two unrelated households residing on different floors in the same stack of an apartment building was reported in Singapore. After cases were conveyed to healthcare facilities and six healthy household contacts were quarantined in their respective apartments, wastewater surveillance was implemented for the entire residential block. SARS-CoV-2 was subsequently detected in wastewaters in an increasing frequency and concentration, despite the absence of confirmed COVID-19 cases, suggesting the presence of fresh case/s in the building. Phone interviews of six residents in quarantine revealed that no one was symptomatic (fever/ respiratory illness). However, when nasopharyngeal swabs from six quarantined residents were tested by PCR tests, one was positive for SARS-CoV-2. The positive case reported episodes of diarrhea and the case's stool sample was also positive for SARS-CoV-2, explaining the SARS-CoV-2 spikes observed in wastewaters. After the case was conveyed to a healthcare facility, wastewaters continued to yield positive signals for five days, though with a decreasing intensity. This was attributed to the return of recovered cases, who had continued to shed the virus. Our findings demonstrate the utility of wastewater surveillance as a non-intrusive tool to monitor high-risk COVID-19 premises, which is able to trigger individual tests for case detection, highlighting a new use-case for wastewater testing.

Wastewater-based surveillance for SARS-CoV-2 has been used for the early warning of transmission or objective trending of the population-level disease prevalence. Here, we describe a new use-case of conducting targeted wastewater surveillance to complement clinical testing for case identification in a small community at risk of transmission. On 2 July 2020, a cluster of COVID-19 cases in two unrelated households residing on different floors in the same stack of an apartment building was reported in Singapore. After cases were conveyed to healthcare facilities and six healthy household contacts were quarantined in their respective apartments, wastewater surveillance was implemented for the entire residential block. SARS-CoV-2 was subsequently detected in wastewaters in an increasing frequency and concentration, despite the absence of confirmed COVID-19 cases, suggesting the presence of fresh case/s in the building. Phone interviews of six residents in quarantine revealed that no one was symptomatic (fever/ respiratory illness).

However, when nasopharyngeal swabs from six quarantined residents were tested by PCR tests, one was positive for SARS-CoV-2. The positive case reported episodes of diarrhea and the case's stool sample was also positive for SARS-CoV-2, explaining the SARS-CoV-2 spikes observed in wastewaters. After the case was conveyed to a healthcare facility, wastewaters continued to yield positive signals for five days, though with a decreasing intensity. This was attributed to the return of recovered cases, who had continued to shed the virus. Our findings demonstrate the utility of wastewater surveillance as a non-intrusive tool to monitor high-risk COVID-19 premises, which is able to trigger individual tests for case detection, highlighting a new use-case for wastewater testing. 

Wastewater-based testing for SARS-CoV-2 has been shown to be a useful COVID-19 surveillance tool (Daughton, 2020) . At wastewater treatment plants, it could provide early warning of transmission or objective trending of the population-level disease prevalence (Thompson et al., 2020) . In countries such as the Netherlands (Medema et al., 2020) , Spain (Randazzo et al., 2020) , France (Wurtzer et al., 2020) and the United States (Nemudryi et al., 2020) , the detection of SARS-CoV-2 RNA at wastewater treatment plants preceded symptomatic cases. Wastewater surveillance has also been used as an additional indicator to provide prevalence trends of SARS-CoV-2 in the sampled communities (National Institute for Public Health and the Environment, 2020; Peccia et al., 2020) . More recently, surveillance at high-density living premises types, such as student hostels, has also been carried out (Chelvan, 2021; Peiser, 2020) .

In Singapore, more than 58,000 COVID-19 cases have been reported from January to December 2020, mainly in community clusters and migrant worker dormitories. Similar to other countries, active wastewater monitoring is also being conducted in Singapore since February 2020 at water reclamation plants for wide area surveillance, and at high density living premises types, such as worker dormitories to provide timely assessment of the COVID-19 situation (Mohan, 2020 ; National Environment Agency, 2020).

On 2 July 2020, the Ministry of Health in Singapore reported a cluster of COVID-19 cases in two unrelated households on different floors of the same apartment building (Goh, 2020) . Six apartment units on each floor of this building share lifts, lift lobbies and stairwell. The first case (index case) was reported on 23 Jun 2020, followed by eight additional cases identified through active clinical surveillance. Six of these were among seven family members who J o u r n a l P r e -p r o o f Journal Pre-proof reside with the index case. Two remaining cases were among seven residents in another unit neighboring the index case ( Figure 1 ). Residents of the two affected units had no known interactions.

All cases were conveyed to healthcare facilities for clinical management and isolation, and household members in the two affected units were placed under home quarantine for 14 days.

Active clinical surveillance that comprised a one-time offer of PCR testing and phone interview for symptoms was conducted among other residents and visitors of the affected building. To complement clinical surveillance, wastewater surveillance was implemented from 4 to 20 July 2020 for continuous monitoring of the building.

Here, we describe a new use-case of wastewater surveillance that facilitated the detection of a new COVID-19 case prior to onset of symptoms in the affected apartment building, demonstrating the utility of wastewater surveillance as a non-intrusive tool to monitor highrisk COVID-19 premises.

Persons under quarantine (PUQs) who resided in the two affected units (one and five PUQs, respectively) were placed on active phone surveillance. Nasopharyngeal PCR swab tests were conducted at the start and end of their quarantine period. Another 152 residents and 25 visitors of the affected section of the building were offered PCR tests and were placed on active phone surveillance and monitored for fever or respiratory symptoms until 12 July 2020. All epidemiological investigations and outbreak containment measures were implemented under the Infectious Diseases Act (Singapore Statutes Online, 2003). 

An aliquot of 45 mL of each hourly composite sample was concentrated using an adapted polyethylene glycol (PEG) precipitation method (Ahmed et al., 2020; World Health Organization, 2003) . Briefly, each sample aliquot was clarified through centrifugation at 4,000 x g for 30 minutes at 4 o C. The supernatant was added to 3.6 g of PEG (8% w/v, polyethylene glycol 8000, Sigma Aldrich) and 0.875 g of sodium chloride (Sigma Aldrich).

The supernatant-PEG mixture was incubated overnight at 4 o C on a rotating shaker at 200 rpm and was subsequently centrifuged at 4,000 x g for 180 minutes at 4 o C. The supernatant was removed, and the virus pellet was suspended in 300 μL of phosphate buffered saline.

This adapted method had a SARS-CoV-2 recovery rate of 59.5 ± 10.4 % and was chosen because of its higher recovery rate in our laboratory settings when compared with other reported virus concentration methods (Ahmed et al., 2020) . The recovery rate for the virus 

SARS-CoV-2 ORF1ab gene (World Health Organization, 2020) and the PMMoV faecal indicator (Gu et al., 2018) were detected in RNA extracted from wastewater samples by using quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) assays described elsewhere (Niu et al., 2020; Zhang et al., 2006) . PMMoV, which is widespread and abundant in human faeces (Kitajima et al., 2018; Rosario et al., 2009) The lowest level of detection for ORF1ab was 10 copies of synthetic SARS-CoV-2 RNA per reaction at an average Cq of 39.36. The respective value for PMMoV target was 150 copies of synthetic RNA per reaction at an average Cq of 39.96. Therefore, evidence of amplification at Cq <40 was considered as a "positive signal" for each target. All tests were performed in triplicates and an average concentration is reported.

The one-time swab tests were done on 110 residents and 13 visitors of the affected building from 1 to 3 July 2020. All tests yielded negative SARS-CoV-2 results. However, SARS-CoV-2 was detected in one of eight wastewater samples collected on the first day of deployment on 4 July 2020 (7,000 RNA copies/L sewage) (Figure 2A ), suggesting active virus shedding in the building. SARS-CoV-2 was detected in wastewaters daily from 4 to 9

July 2020, with frequent spikes ranging from 600 to 246,700 RNA copies/L sewage ( Figure   2A ) CoV-2 RNA was detected in wastewaters more than three days before the case recalled diarrheal, fever and respiratory symptoms, suggesting that viral shedding in faecal material could have started in the pre-symptomatic period.

The index case (Case 1) returned from the healthcare facility on 8 July 2020 after clinical recovery. Therefore, wastewater signals detected on 9 July 2020 could be either due to viral shedding from the newly identified case (Case 10) or from the returned index case. However, SARS-CoV-2 levels declined after the newly identified case was conveyed to a healthcare facility (1,100 -39,400 to 600 -1,700 RNA copies/L sewage from 10 to 13 July 2020) ( Figure 2B ), suggesting that subsequent viral shedding was from the recovered index case, rather than from another additional new case/s. This contrasts to the frequent spikes in virus RNA levels observed from 4 to 9 July 2020, before the new case was identified.

Interestingly, SARS-CoV-2 levels increased again during 15 -16 July 2020 (3,900 -28,300

RNA copies/L sewage) and was attributed to two recovered cases who returned on 14 July 2020 ( Figure 2B ). SARS-CoV-2 levels were not detected from the evening of 19 July 2020, corroborating the viral shedding pattern of recovered cases and the cessation of active virus transmission in the building.

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

This report describes a new use-case for wastewater surveillance, in which monitoring of a relatively small community at high risk of COVID-19 provided actionable data that informed operational decisions. Positive and intensifying SARS-CoV-2 signals in wastewater triggered intrusive individual tests for case identification, while negative or weakening signals assured the gradual cessation of transmission. In contrast to the widely reported, regional-based surveillance at wastewater treatment plants implemented for situational assessment (La Rosa et al., 2020; Prado et al., 2021) , this targeted approach of wastewater surveillance complemented the cost-and logistics-intensive individual testing to facilitate early identification of a COVID-19 case.

Detection of virus RNA in wastewaters before the onset of symptoms in the present study supports the notion that faecal material can be positive for SARS-CoV-2, even when cases are asymptomatic (Ahmad et al., 2020; Tang et al., 2020) . It is now known that SARS-CoV-2 viral shedding can persist longer in stool samples than in nasopharyngeal swabs (Gupta et al., 2020) . Taken together, this suggests that stool samples have a longer detection window for SARS-CoV-2 than other commonly-tested biological samples, underscoring the utility of wastewater testing as a COVID-19 surveillance strategy.

The present study also revealed the importance of longitudinal wastewater surveillance for the continuous monitoring of SARS-CoV-2 in the community. Single time point detection of SARS-CoV-2 in wastewaters provided limited information on whether a positive signal originates due to viral shedding by newly-emerging cases or from a recovered case. In contrast, continued monitoring and trend analyses could reveal whether positive signals occur due to virus shedding either by a new or a recovered case. Our findings also emphasized that the contribution of residual virus shedding from recovered cases must be accounted for when J o u r n a l P r e -p r o o f modelling wastewater surveillance-based estimates of COVID-19 prevalence in affected communities.

It was a challenge to discern whether virus signals were due to new or recovered cases, Despite demonstrating a new use-case for wastewater surveillance, the approach described in the present study might be resource-intensive and/or limited by the access to suitable sampling sites to capture the targeted population

 This study highlights a new use-case for non-intrusive wastewater testing at high-risk sites that facilitates case identification with minimal inconvenience to the community.

 The study demonstrated that virus shedding by even a single case could be detected in wastewater from a small community. 

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The authors would like to thank Ms Quek Hui Leng for her administrative support as well as Janelle Thompson, Stefan Wuerst, Karina Gin and Eric Alm for their useful discussions.Funding/Support: This study was internally funded by the National Environment Agency.J o u r n a l P r e -p r o o f Journal Pre-proof

The authors have no conflicts of interest to declare.J o u r n a l P r e -p r o o f