key: cord-0840132-5snu32y9 authors: Deng, Yu; Xu, Xiaoqing; Zheng, Xiawan; Ding, Jiahui; Li, Shuxian; Chui, Ho-kwong; Wong, Tsz-kin; Poon, Leo L.M.; Zhang, Tong title: Use of sewage surveillance for COVID-19 to guide public health response: A case study in Hong Kong date: 2022-01-19 journal: Sci Total Environ DOI: 10.1016/j.scitotenv.2022.153250 sha: f119e4e4ec13641877ba5714ca709a166052ad6b doc_id: 840132 cord_uid: 5snu32y9 Sewage surveillance could help develop proactive response to the Coronavirus Disease 2019 (COVID-19) pandemic, but currently there are limited reports about examples in practical exercises. Here, we report a use case of intensified sewage surveillance to initiate public health action to thwart a looming Delta variant outbreak in Hong Kong. On 21 June 2021, albeit under basically contained COVID-19 situation in Hong Kong, routine sewage surveillance identified a high viral load of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a sewage sample from one site covering over 33,000 population, suggesting infected cases living in the respective sewershed. The use of a newly developed method based on allele-specific real-time quantitative polymerase chain reaction (AS RT-qPCR) served to alert the first documentation of the Delta variant in local community sewage three days before the case was confirmed to be a Delta variant carrier. Intensified sewage surveillance was then triggered. Targeted upstream sampling at sub-sewershed areas pinpointed the source of positive viral signal across spatial scales from sewershed to building level, and assisted in determining the specific area for issuing a compulsory testing order for individuals on 23 June 2021. A person who lived in a building with the positive result of sewage testing was confirmed to be infected with COVID-19 on 24 June 2021. Viral genome sequences determined from the sewage sample were compared to those from the clinic specimens of the matched patient, and confirmed that the person was the source of the positive SARS-CoV-2 signal in the sewage sample. This study could help build confidences for public health agencies in using the sewage surveillance in their own communities. Sewage surveillance; Public health intervention; Allele-specific RT-qPCR; Delta variant; Targeted upstream sampling During the COVID-19 pandemic, accurate use of diagnostic tests in high volumes and quantities as well as the rapid response to the testing results help implement proactive public health response 1 Long-lasting shedding of SARS-CoV-2 in the human excreta was identified regardless of disease severity 4, 5 . The value of integrating sewage surveillance into the management system of COVID-19 outbreaks is high, especially for early identification of asymptomatic or presymptomatic individuals at the community-level in a scalable, cost-effective and noninvasive way 6 . testing data for assisting the decision making process, an interactive online dashboard was built to integrate data from multiple sources based on Geographic Information Systems (GIS), including sewage testing data, geographical data of the sampling site sewershed, population survey data of individual sewersheds, georeferenced clinic case record, COVID-19 hospitals, and designated quarantine hotels and centres. The use of sewage surveillance in Hong Kong started from pilot studies for target vulnerable populations at residential care homes when the fourth COVID-19 wave hit Hong Kong in November 2020, geared to scale implementation at ad hoc sites of residential buildings and city blocks with clusters of infections since December 2020, to sentinel monitoring at stationary sites across for early warning after the fourth wave decelerated in March 2021. Total sampling points of both stationary sites and ad hoc sites as of August 2021 exceeded over 1,500 ( Figure 1 ). The objectives of this study were to exemplify how systematic sewage surveillance identified a single building with only one COVID-19 patient as the source of viral signal in sewage from a sewershed of 33,000 people and the way in using a new workflow to help rapidly identify the Delta variant in sewage samples, with the aim to provide near real-time results for decision making in assisting the successful containment of a silent transmission event of SARS-CoV-2 in the local community in Hong Kong. J o u r n a l P r e -p r o o f Journal Pre-proof All sewage samples were collected from manholes and taken at 15-minute interval during 3 hours in the morning peak (8 am to 11 am). The adopted sampling strategy using 3-hour composite sampling spanning the morning 8 ~11 am could allow for timely processing of the sewage samples within 24 hours after the sampling event as well as minimisation of dilution effect of the viral signals caused by longer sampling time for the composite sample. Sewage samples were regularly collected from 112 stationary sampling sites across various districts in Hong Kong. The site TP site 9A was one of the stationary site located in the Tai Po (TP) district, which covered a serving population of 33,000. Samples were delivered to the laboratory on ice. 50 mL samples were inactivated at 60 C for 30 minutes and were firstly centrifuged at 4,750 g at 4 C for 30 minutes, and then 30 mL supernatant was used for further ultracentrifugation at 150,000 g at 4 C for 1 hour (Beckman Coulter, Indianapolis, IN). Finally, the concentrated pellet from the last step was resuspended with ~ 200 µL PBS for RNA extraction. RNA was extracted using QIAamp Viral RNA Kits (Qiagen) according to the manufacturer's instruction with a final elution volume of 50 µL. The primers and probes were designed using Clone Manager 8.0 (Sci-Ed Software), following the principle of allele-specific RT-qPCR to place the mutation in 3' end of forward or reverse primer. We designed two forward primer sets respectively targeting wild type and mutation type for each genomic site, and there was a probe and reverse primer for each target site. In addition, we introduced an artificial mismatch close to 3' end of forward primer to boost the discrimination between the wild type and the mutant. The parameters including J o u r n a l P r e -p r o o f Journal Pre-proof hairpin, self-dimerization, and heterodimerization of all primer and probe sequences were analyzed using Clone Manager 8.0 (Sci-Ed Software). Detailed information of the primers and probes designed were included in Table S1 . All primers and probes were synthesized by BGI. To verify the performance of designed primers and probes for sensitivity and specificity, we synthesized three plasmids from BGI respectively carrying the wild type of the spike gene, the mutant type with L452R, N501Y and P681R mutation sites, and the mutant type with T478K. They were referred to as the wild type (WT) and the mutant type (MT). All fragments were cloned to a commercial PMV vector. All synthesized plasmids were dissolved into DEPC-treated water and quantified Qubit dsDNA HS assay kit (ThermoFisher, USA). The copy number was obtained by the length and Avogadro's number. The standard curves were generated by tenfold dilution of the plasmids from 10 5 to 10 0 copy µL -1 . The final concentration of SARS-CoV-2 in raw sewage, as measured in copy per L, was derived by dividing the viral concentration in one RNA extraction by the volume of the tested raw sewage sample (30 mL). One RNA extraction was the total RNA volume (i.e., 50 µL) extracted from the concentrated sample obtained from the 30 mL raw sewage sample. And the viral concentration in one RNA extraction was calculated by multiplying the measured concentration from RT-qPCR reaction by the ratio of the RNA volume for one RNA extraction (i.e., 50 µL) to that used for one RT-qPCR reaction (4 µL). primer-probe set that has a higher average viral concentration for duplicated RT-qPCR reactions calculated using the standard curves of N1-or E-carrying plasmids. The plasmid carrying N1 target or E target was synthesized commercially by BGI. The concentration of plasmid was obtained using Qubit dsDNA HS assay kit (ThermoFisher, USA) and the copy number was calculated by the length and Avogadro's number. In each batch of RT-qPCR, the standard curves for N1 and E were freshly made by serial tenfold dilution of the plasmid ranging from 10 to 10 6 copy µL -1 to quantify the copy number of SARS-CoV-2 in positive sewage samples. For the variant detection, we used specifically designed primer sets. The One-step RT-qPCR reactions were set up in a final volume of 20 µL, using 4 µL template, 5 µL TaqMan Fast (%) = 100 * (10 − 1 − 1). We applied targeted sequencing using Nanopore and NovaSeq platforms in this study. ARTIC amplicons (~400 bp) of TP site 9A sample were prepared according to the ARTIC V3 protocol the PCR for sequencing with some modifications, specifically, we added 10 µL of cDNA template instead of 2.5 µL. The purified amplicons were subjected to Next-Generation sequencing (NGS, NovaSeq 6000, Novogene) and Nanopore sequencing. For nanopore sequencing, we used ONT ligation Kit (SQK-LSK109) following the manufacturer's protocol. The library was run into a flowcell (FLO-MIN106) and sequenced on a GridION X5 device. The amplicon-based sequencing for the patient was performed via Illumina platform using method as previously reported 23 . The resulting reads for NGS were aligned to the reference genome sequence of a SARS-CoV- We used GraphPad Prism (GraphPad Software, San Diego, CA, USA) and Microsoft Excel for linear regression and other statistical analyses. The sewage surveillance system in Hong Kong now serves as a management tool for proactive preparedness and responses to the COVID-19 pandemic, with adaptive sampling J o u r n a l P r e -p r o o f strategy for providing an early warning signal, monitoring different phases of pandemic progression to meet the public health needs, as well as real-time reporting to assist the decision making process. Once a sewage sample is tested as "positive" for SARS-CoV-2, the health authority will consider relevant clinical factors to decide public health interventions on the need, such as a compulsory testing notice or a restriction-testing declaration, to be conducted for the concerned population to identify the hidden cases. For the ongoing routine sewage surveillance in Hong Kong, sewage samples are daily collected and analysed for the presence of SARS-CoV-2. As of June 2021, around 220 sewage samples were collected and tested weekly from 112 stationary sampling sites that covered sewage discharged from nearly 5.3 million people (70% of the total population). This provides broad monitoring on the overall COVID-19 condition across the territory in a proactive manner. We implemented a rapid testing workflow for detecting SARS-CoV-2 Delta variant. This The analytical sensitivity and specificity of the AS RT-qPCR panel were evaluated using synthetic DNA constructs of four mutations and their opposite genotypes ( Figure S1 ). Based on the testing of individual genome targets using the designed pair of primer-probe sets, we showed that the AS RT-qPCR panel reliably detected all eight genotype targets at 10-100 viral DNA copy per reaction with amplification efficiencies above 90%. The analytic specificity was determined by the level of cross-reactivity for the non-target genotype. Crossreactivity was minimal for N501Y and P681R mutation types and their respective wild types, and was only observed for targeted DNA constructs at > 10 4 viral DNA copy per reaction. A similar level of cross-reactivity was also observed for the T478K and L452R mutation types at > 10 4 and > 10 3 viral DNA copy per reaction, respectively. But the primer-probe sets for the T478 and L452 wild types were less specific, with observed cross-reactivity at > 10 2 viral DNA copy per reaction. Still, the T478 and L452 primer-probe sets were discriminated against the non-target mutation type sequences. In order to cope with the rapid assessment of public health threats needed for the decision making process, the first positive sewage sample collected from the site TP site 9A on 21 June 2021 was tested using the new workflow. The L452R mutation type and the N501 wild In In this study, we showed an example on the use of sewage surveillance for COVID-19 to address the operationalization needs and the decision making timeline for a public health It should be noted that, one limitation for the AS-qPCR method used in this study was the cross-reactivity observed for the primer-probe sets designed for the mutation sites of T478K and L452R. Future work is needed to improve the specificity by optimizing the design of the primer sequence and the RT-qPCR conditions. 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